Regulated expression of antigen and/or regulated attenuation to enhance vaccine immunogenicity and/or safety

ABSTRACT

The invention relates to compositions and methods for making and using recombinant bacteria that are capable of regulated attenuation and/or regulated expression of one or more antigens of interest.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/850,957, filed on Dec. 21, 2017, now abandoned which, in turn, is acontinuation of U.S. patent application Ser. No. 15/040,005, filed onFeb. 10, 2016 (now U.S. Pat. No. 9,885,051, issued Feb. 6, 2018), which,in turn, is a continuation of U.S. patent application Ser. No.13/789,665, filed on Mar. 7, 2013 (now U.S. Pat. No. 9,297,015, issuedMar. 29, 2016), which, in turn, is a continuation of U.S. patentapplication Ser. No. 12/615,872, filed on Nov. 10, 2009 (now U.S. Pat.No. 8,445,254, issued on May 21, 2013) which, in turn, is acontinuation-in-part of PCT/US2008/063293, filed May 9, 2008, which inturn claims priority to U.S. Provisional Patent Application No.60/917,313, filed on May 10, 2007, the entire contents of each of whichare expressly incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 5, 2019, isnamed 127225-00106-SequenceListing.txt and is 22,461 bytes in size.

GOVERNMENTAL RIGHTS

This invention was made with government support under DE006669,AI056289, AI057885, AI024533 awarded by the National Institutes ofHealth and 2003-35204-13748 and 99-35204-8572 awarded by the Departmentof Agriculture. The government has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to compositions and methods for making and usingrecombinant bacteria that are capable of regulated attenuation and/orregulated expression of at least one nucleic acid encoding an antigen ofinterest.

BACKGROUND OF THE INVENTION

Recombinant microorganisms have widespread utility and importance. Oneuse of these microorganisms is as live vaccines to produce an immuneresponse. Live vaccines are most effective when they produce high levelsof antigen. However, the synthesis of a recombinant antigen encoded by ahighly expressed nucleic acid sequence may be deleterious to themicroorganism. Because of this, regulated (as opposed to constitutive)expression systems have been identified and utilized where therecombinant nucleic acid sequence of interest is operably linked tocontrol elements that allow expression of significant amounts of therecombinant nucleic acid sequence only when it is induced, derepressedor activated. Examples include the cspA nucleic acid sequence promoter,the phoA nucleic acid sequence promoter, P_(BAD) (in an araC-P_(BAD)system), the trp promoter, the tac promoter, the trc promoter, A P_(L),P22 P_(R), mal promoters, rha promoter, xyl promoter, and the lacpromoter. These promoters may mediate transcription at low temperature,at low phosphate levels, in the presence of arabinose, in the presenceof at low tryptophan levels, in the presence of rhamanose, in thepresence of xylose, and in the presence of lactose (or other lacinducers).

When the recombinant microorganism is used as a vertebrate live vaccine,certain considerations must be taken into account. To provide a benefitbeyond that of a nonliving vaccine, the live vaccine microorganism mustattach to, invade, and survive in lymphoid tissues of the vertebrate andexpose these immune effector sites to antigen for an extended period oftime. Through this continual stimulation, the vertebrate's immune systembecomes more highly reactive to the antigen than with a nonlivingvaccine. Therefore, preferred live vaccines are attenuated pathogens ofthe vertebrate, particularly pathogens that colonize the gut-associatedlymphoid tissue (GALT), nasopharynx-associated lymphoid tissue (NALT) orbronchial-associated lymphoid tissue (BALT). An additional advantage ofthese attenuated pathogens over nonliving vaccines is that thesepathogens have elaborate mechanisms to gain access to lymphoid tissues,and thus efficient exposure to the vertebrate's immune system can beexpected. In contrast, nonliving vaccines will only provide an immunestimulus if the vaccine is passively exposed to the immune system, or ifhost mechanisms bring the vaccine to the immune system.

Pathogenic bacteria may be attenuated by mutation so that uponinfection, host disease symptomology is not elicited. Most means ofattenuation, however, make live vaccine strains more suceptable thanwild-type strains to environmental stresses encountered afterinoculation into the animal or human host. Consequently, fewer bacteriasurvive to colonize the GALT, NALT and/or BALT with a reduction ineffective immunogenicity of the vaccine. Thus these attenuationmechanisms hyperattenuate the vaccine, precluding the candidate vaccinefrom either reaching or persisting in lymphoid tissues to a sufficientextent or duration to permit induction of a protective immune responseagainst the wild-type pathogen of interest. Thus, there is a need in theart for methods of regulating the expression of the attenuatedphenotype. This allows the live vaccine strain to display abilitiessimilar to a wild-type virulent parental pathogen in order tosuccessfully colonize effector lymphoid tissues prior to the display andimposition of the full attenuated phenotype to preclude induction ofdisease symptoms.

Since immune responses induced against foreign antigens are proportionalto the levels of antigen synthesized by the recombinant attenuatedbacterial vaccine (1,3), the placement of the nucleic acid sequence forthe foreign antigen on a multi-copy plasmid vector is much preferable tothe insertion of the nucleic acid sequence for the foreign antigen intothe chromosome of the attenuated bacterial vaccine vector. This isbecause the level of foreign antigen synthesis is generally proportionalto the number of copies of the nucleic acid sequence for the foreignantigen expressed within the attenuated bacterial host.

Since plasmid-containing recombinant attenuated bacterial vaccinesoverproduce large amounts of antigen that provide no advantage to thevaccine, the plasmid vectors are often lost over time afterimmunization. In many cases, ten percent or less of the recombinantattenuated bacterial vaccine isolated from the immunized vertebrateretains the plasmid after three or four days. When this plasmid lossoccurs, the immune response is directed more against the attenuatedbacterial host vaccine itself rather than against the expressed foreignantigen.

As stated above, the level of immune response to a foreign antigen isgenerally proportional to the level of expression of the nucleic acidsequence encoding the antigen. Encoding the protective antigen on aplasmid vector is important in maximizing the production of thatprotective antigen, which is very much correlated with the ability toinduce high level protective immune responses by production of mucosaland systemic antibodies against the protective antigen. Unfortunately,overexpression of nucleic acid encoding a foreign antigen is often toxicsuch that it reduces the rate of growth and therefore the ability of theattenuated bacterial vaccine to colonize lymphoid tissues. As aconsequence, the ultimate immunogenicity is sharply diminished. For thisreason, it is necessary to balance the ability of the vaccine tocolonize and grow in lymphoid tissues with the ability to synthesize theforeign antigen.

SUMMARY OF THE INVENTION

One aspect of the present invention encompasses a recombinant bacteriumcapable of the regulated expression of at least one nucleic acidsequence encoding an antigen of interest, and capable of regulatedattenuation. The bacterium comprises at least one chromosomallyintegrated nucleic acid sequence encoding a repressor operably linked toa regulatable promoter and a vector comprising a nucleic acid sequenceencoding at least one antigen of interest operably linked to a promoterregulated by the repressor, such that the expression of the nucleic acidsequence encoding the antigen is repressed during in vitro growth of thebacterium, but the bacterium is capable of high level expression of thenucleic acid sequence encoding the antigen in a host. The bacteriumfurther comprises a regulatable promoter chromosomally integrated so asto replace the native promoter of, and be operably-linked to, at leastone nucleic acid sequence of an attenuation protein.

Another aspect of the invention encompasses a recombinant bacteriumcapable of regulated expression of at least one nucleic acid sequenceencoding an antigen of interest. The bacterium comprises at least onechromosomally integrated nucleic acid sequence encoding a repressoroperably linked to a regulatable promoter, wherein the nucleic acidsequence encoding the repressor and/or promoter have been modified fromthe wild-type nucleic acid sequence so as to optimize the expressionlevel of the nucleic acid sequence encoding the repressor, and a vectorcomprising at least one nucleic acid sequence encoding an antigen ofinterest operably linked to a promoter regulated by the repressor, suchthat the expression of the nucleic acid sequence encoding the antigen isrepressed during in vitro growth of the bacterium, but the bacterium iscapable of high level expression of the nucleic acid sequence encodingthe antigen in a host.

Yet another aspect of the invention encompasses a recombinant bacteriumcapable of regulated attenuation. The bacterium comprises a modifiedregulatable promoter chromosomally integrated so as to replace thenative promoter of, and be operably linked to, at least one nucleic acidsequence encoding an attenuation protein.

Other aspects and iterations of the invention are described morethoroughly below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts an illustration of various deletion-insertion mutationsof relA and various embodiments of lacl optimization. The schematicshows the deletion of 2247 bp (relA-12 to relA 2235/2235) and insertionof 2429 bp of araC P_(BAD) lacl TT. The optimized sequences ofΔrelA196::araC P_(BAD) lacl TT (SEQ ID NO:1), ΔrelA197::araC P_(BAD)lacl TT (SEQ ID NO:2), and ΔrelA198::araC P_(BAD) (SEQ ID NO:3) lacl* TTshow variations of SD sequences and start codons. lacl*: codonoptimized.

FIG. 2 depicts an illustration of a chromosomal map of thedeletion-insertion mutation of endA and various embodiments of lacloptimization. The schematic shows the deletion of 719 bp (endA-11 toendA 708/708) and insertion of 2429 bp of araC P_(BAD) lacl TT. Theoptimized sequences of ΔendA19::araC P_(BAD) lacl TT (SEQ ID NO:4),ΔendA20::araC P_(BAD) lacl TT (SEQ ID NO:5), and ΔendA21::araC P_(BAD)lacl* TT (SEQ ID NO:6) show variations of SD sequences and start codons.

FIG. 3A and FIG. 3B depict an illustration of nucleic acid sequencemodification analyses of SD-lacl in ΔendA19 and ΔrelA196 (SEQ ID NO:7),SD-lacl in ΔendA20 and ΔrelA197 (SEQ ID NO:8), and SD-lacl in ΔendA21and ΔrelA198 (SEQ ID NO:9). The amino acid sequence of Lacl is included(SEQ ID NO:10).

FIG. 4 depicts an illustration of a chromosomal map of variousdeletion-insertion mutations of asdA and various embodiments of c2optimization. The schematic shows the deletion of 1104 bp from the asdnucleic acid sequence (1 to 1104 not including TAG stop codon) and theinsertion of 1989 bp of c2 P_(BAD) araC TT. The optimized sequences ofΔasdA18::TT araC P_(BAD) c2 (SEQ ID NO: 1), ΔasdA20::TT araC P_(BAD) c2*(SEQ ID NO:12), ΔasdA21::TT araC P_(BAD) c2* (SEQ ID NO:13), andΔasdA27::TT araC P_(BAD*) c2** (SEQ ID NO:14) show variations of SDsequences and start codons. P_(BAD*): the −10 sequence is improved fromTACTGT to TATAAT; c2*: SD-codon optimized; c2**: SD-codon optimized andsecond codon is modified to AAA from AAT.

FIG. 5 depicts an illustration of c2 original sequence (SEQ ID NO:15)aligned with the optimized sequence (SEQ ID NO:16). The amino acidsequence of the optimized sequence is shown (SEQ ID NO:17). According tothe actual DNA sequence data, the amino acid at position 49 has beenaltered to serine (S) from aspartic acid (N) (AAC to AGC).

FIG. 6 depicts an illustration of the original (SEQ ID NO:18) and themodified (SEQ ID NO:19) P_(BAD) region and the original (SEQ ID NO:20)and modified (SEQ ID NO:21) c2 sequence. The original C2 amino acidsequence (SEQ ID NO:22) is compared to the optimized sequence (SEQ IDNO:23).

FIG. 7 depicts an illustration of a chromosomal map of thedeletion-insertion mutation of the crp promoter region. The schematicshows the deletion of the crp promoter region (−15 to −109) and theinsertion of 1335 bp of TT araC P_(BAD). TT: T4 iplll TranscriptionTerminator

FIG. 8 depicts an illustration of a chromosomal map of variousdeletion-insertion mutations of the fur promoter region. The schematicshows the deletion of the fur promoter region (−15 to −253; includingFur consensus, Crp binding, and OxyR binding sites) and the insertion of1335 bp of P_(BAD) araC TT. The optimized sequences of ΔP_(fur33)::TTaraC P_(BAD) fur (SEQ ID NO:24), ΔP_(fur77)::TT araC P_(BAD) fur (SEQ IDNO:25), and ΔP_(fur81)::TT araC P_(BAD) fur (SEQ ID NO:26) showvariations of SD sequences and start codons.

FIG. 9 depicts an illustration of a chromosomal map of variousdeletion-insertion mutations of the phoPQ promoter region. The schematicshows the deletion of the phoPQ promoter region (−12 to −109) and theinsertion of 1335 bp of P_(BAD) araC TT. The optimized sequences ofΔP_(phoPQ107)::TT araC P_(BAD) phoPQ (SEQ ID NO:27), ΔP_(phoPQ173)::TTaraC P_(BAD) phoPQ (SEQ ID NO:28), and ΔP_(phoPQ177)::TT araC P_(BAD)phoPQ (SEQ ID NO:29), ΔP_(phoPQ174)::TT araC P_(BAD) phoPQ (SEQ IDNO:30), ΔP_(phoPQ175)::TT araC P_(BAD) ΔGAG-phoPQ (SEQ ID NO:31), andΔP_(phoPQ176)::TT araC P_(BAD) ΩCTC-phoPQ (SEQ ID NO:32) show variationsof SD sequences and start codons.

FIG. 10 depicts an illustration of a chromosomal map of thedeletion-insertion mutation of the rpoS promoter region. The schematicshows the deletion of 36 bp of the rpoS promoter region (rpoS-13 to -48)and the insertion of 1335 bp of P_(BAD) araC TT.

FIG. 11 depicts an illustration of a chromosomal map of thedeletion-insertion mutation of the murA promoter region. The schematicshows the deletion of 42 bp between murA and yrbA and the insertion of1335 bp of P_(BAD) araC TT.

FIG. 12 depicts an illustration of the pYA3493 plasmid.

FIG. 13 depicts an illustration of the pYA3634 plasmid.

FIG. 14 depicts an illustration of the pYA3681 plasmid.

FIG. 15 depicts a photograph and a graph showing results from a westernblot analysis on Salmonella UK-1 ΔrelA::araC P_(BAD) lacl (GTG vs ATG vsATG codon) TT mutations using rabbit Lacl antiserum.

FIG. 16 depicts a photograph showing results from a western blotanalysis on Salmonella UK-1 ΔasdA::TT araC P_(BAD) c2 mutants usingpolyclonal C2 antiserum.

FIG. 17 depicts an illustration of a chromosomal map of thedeletion-insertion mutation of the araC P_(BAD) region and the DNAsequence of P22 P_(R) araB region (SEQ ID NO: 33). The schematic showsthe deletion of 1169 bp including 846 bp of araC and 323 bp of theP_(BAD) region and the insertion of 91 bp of P22 P_(R) sequence.

FIG. 18 depicts a graph showing survival after S. pneumoniae challenge.

FIG. 19A, FIG. 19B and FIG. 19C depict principle of regulated delayedexpression and constructions of strains with ΔrelA::araC P_(BAD) lacl TTcassette. (FIG. 19A) Principle of regulated delayed expression. Thissystem includes a chromosomal repressor gene, lacl, expressed from thearabinose-regulated araC P_(BAD) promoter. Lacl regulates expressionfrom a plasmid promoter, P_(trc) that directs antigen synthesis. In thepresence of arabinose, Lacl is produced which binds to P_(trc), blockingantigen encoding sequence expression. In host tissues, an arabinose poorenvironment, the concentration of Lacl will decrease with each celldivision allowing increased antigen synthesis, thus inducing an immuneresponse. (FIG. 19B) Strains with different levels of Lacl encodingsequence expression due to altered SD-sequence, start codon and codonusage in the lacl gene were inserted into the relA gene site of theSalmonella genome. The deletion-insertion deleted 2247 bp in relA(relA-12 to relA 2235) and inserted 2429 bp of araC P_(BAD) lacl TTcassette. These mutations were also introduced into strains with theΔpabA ΔpabB ΔasdA ΔaraBAD genotype to provide attenuation, selection fora balanced-lethal vector (Asd⁺) and to block arabinose metabolism. (FIG.19C) Western blot analysis of ΔrelA::araC P_(BAD) lacl (GTG vs. ATG vs.ATG codon optimized) mutations using rabbit anti-Lacl antiserum. Thestrains were grown in 3XD media with different concentrations ofarabinose. The samples were normalized by cell number. Densitometry wasmeasured by Quantityone software. The number shows the relativedensitometry.

FIG. 20A and FIG. 20B depict two graphs showing that higher expressionof Lacl encoding sequence does not change growth. The strains were grownin LB media with 0% (dashed line) and 0.2% arabinose (solid line). TheOD₆₀₀ was measured at 40 min intervals. *, χ9097, ▴, χ9095, ●, χ9101, ▪,χ9241 (FIG. 20A) Strains without antigen encoding sequence expressionplasmid. DAP was included in the growth medium for these strains. (FIG.20B) Strains with antigen encoding sequence expression plasmid pYA4088.

FIG. 21A, FIG. 21B and FIG. 21C depict a series of graphs illustratingthe effect of arabinose on gfp expression and the kinetics of Lacldecrease and PspA antigen increase. (FIG. 21A) Different repressionlevels of GFP synthesis achieved by varied concentrations of arabinose.These strains have plasmid pYA4090 expressing GFP with P_(trc) promoter.Overnight nutrient broth cultures grown without arabinose were diluted1:100 into pre-warmed nutrient broth with 2%, 0.2%, 0.02%, 0.002% and 0%arabinose. When OD₆₀₀ reached 0.4, samples were diluted 1:100 into PBSand subjected to FACS analysis. (FIG. 21B) Kinetics of GFP synthesis.Overnight cultures with different concentrations of arabinose werediluted 1:100 into pre-warmed nutrient broth with arabinose; grown toOD600 of 0.6, and diluted 1:100 into the same pre-warmed media withoutarabinose. The process was repeated two more times. At each time pointof OD₆₀₀ about 0.6, samples were diluted 1:200 into PBS and subjected toFACS analysis. (FIG. 21C) Kinetics of Lacl decrease and PspA antigenincrease. Overnight cultures with 0.2% arabinose were diluted 1:100 intopre-warmed LB media with 0.2% arabinose, grown to an OD₆₀₀ of 0.6, andthen diluted 1:10 into pre-warmed LB media without arabinose. Theprocess was repeated four times. At each time point of OD₆₀₀ about 0.6,equal numbers of samples were taken for western blot analysis usinganti-Lacl and anti-PspA antisera. The densitometry was measured byQuantityone software.

FIG. 22 depicts a graph showing the stability of Lacl protein indifferent strains. XL1-Blue (lacl^(q) E. coli) was grown in LB media.Strains χ8990, χ9080 and χ9226 were grown in 3XD media with 0.2%arabinose to OD₆₀₀ of 0.6 and washed 2 times with 3XD media withoutarabinose. Chloramphenicol were added to 50 μg/ml. Samples were takenbefore washing (pre 0), just after adding chloramphenicol (0), and at 1,2, 4, 6, 8, 24 h and subjected to western blot analysis. The sampleswere normalized by cell number. The densitometry was measured byQuantityone software.

FIG. 23A and FIG. 23B depict a graph showing antibody titers against LPSand PspA. These strains were transformed with plasmid pYA4088 expressingS. pneumoniae PspA Rx1 antigen and vector plasmid pYA3493. Strains weregrown with 0.2% arabinose in LB medium before inoculating mice. (FIG.23A) anti-LPS antibody titers; (FIG. 23B) anti-PspA antibody titers.

FIG. 24 depicts a graph showing a survival curve after challenge withvirulent S. pneumonia WU2 strain. Female BALB/c mice were immunized witha single dose of the indicated strains grown in LB containing 0.2%arabinose. Eight weeks after immunization, mice were challenged with 250LD₅₀ of virulent S. pneumoniae WU2. All mice immunized withPspA-expressing strains were significantly protected (p<0.05). Micevaccinated with strain χ9101 (pYA4088) showed significantly higherprotection than mice vaccinated with the other strains (p<0.05). Theremaining vaccinated groups were not significantly different from eachother (p>0.05).

FIG. 25A, FIG. 25B, FIG. 25C and FIG. 25D depict schematics illustratingdifferent deletion-insertion mutations resulting in arabinose-regulatedvirulence trait. (FIG. 25A) The schematic shows the deletion of the furpromoter region (−15 to −253; including Fur consenus, Crp binding, andOxyR binding sites) and the insertion of 1335 bp of P_(BAD) araC TT tocreate the ΔP_(fur33)::TT araC P_(BAD) fur insertion-deletion mutation.(FIG. 25B) The schematic shows the deletion of the phoPQ promoter region(−12 to −109) and the insertion of 1335 bp of P_(BAD) araC TT to createthe ΔP_(phoPQ107)::TT araC P_(BAD) phoPQ deletion-insertion mutation.(FIG. 25C) The schematic shows the deletion of 36 bp of the rpoSpromoter region (−13 to −48) and the insertion of 1335 bp of P_(BAD)araC TT to create the ΔP_(rpoS183)::TT araC P_(BAD) rpoSdeletion-insertion mutation. (FIG. 25D) The schematic shows the deletionof the crp promoter region (−15 to −109) and the insertion of 1335 bp ofTT araC P_(BAD) to create the ΔP_(crp527)::TT araC P_(BAD) crpinsertion-mutation.

FIG. 26A, FIG. 26B, FIG. 26C and FIG. 26D depict several photographsillustrating the phenotypes of strains with deletion-insertion mutationsto enable arabinose-dependent expression of virulence traits. (FIG. 26A)χ9021 with ΔP_(crp527)::TT araC P_(BAD) crp mutation streaked onMacConkey maltose agar without and with 0.2 percent arabinose. (FIG.26B) χ8848 with ΔP_(fur33)::TT araC P_(BAD) fur and χ9107 withΔP_(fur33)::TT araC P_(BAD) fur and ΔP_(crp527)::TT araC P_(BAD) crpmutations spotted on CAS agar plates without and with 0.2 percentarabinose to visualize siderophore production. (FIG. 26C) χ8918 withΔP_(phoPQ107)::TT araC P_(BAD) phoPQ and χ9108 with ΔP_(phoPQ107)::TTaraC P_(BAD) phoPQ and ΔP_(crp527)::TT araC P_(BAD) crp mutationsstreaked on X-P plates without and with 0.2 percent arabinose to revealacid phosphatase activity. (FIG. 26D) χ8956 with ΔP_(rpoS183)::TT araCP_(BAD) rpoS and χ9064 with ΔP_(rpoS183)::TT araC P_(BAD) rpoS andΔP_(crp527)::TT araC P_(BAD) crp mutations streaked onglycogen-indicator agar without and with 0.2 percent arabinose andsprayed with iodine indicator solution.

FIG. 27A and FIG. 27B depict an illustration of deletion mutationsprecluding breakdown of arabinose and enhancing retention of arabinosetaken up by bacterial cells. (FIG. 27A) The schematic shows the deletionof a total of 4110 bp (araB₂ to araD₊₅₂) and the insertion of 22 bp ofSD, NcoI and PmeI at araB2 to create the ΔaraBAD23 mutation. (FIG. 27B)The schematic shows the deletion of a total of 1432 bp (araE−5 toaraE+8) to create the ΔaraE25 mutation.

FIG. 28 depicts several photographs illustrating the stability of Crp,Fur, RpoS and PhoP proteins during incubation of cultures induced forsynthesis of these proteins prior to addition of 30 and 200 μgchloramphenicol/ml of culture. Rabbit antibodies raised againstHis-tagged Crp, Fur and PhoP were used for western blot analyses. Mousemonoclonal antibodies for RpoS was purchased from Neoclone. χ9021(ΔP_(crp527)), χ8848 (ΔP_(fur33)), χ8956 (ΔP_(rpoS183)) and χ8918(ΔP_(phoPQ107)) were grown in LB broth with 0.2 percent arabinose forthese studies.

FIG. 29 depicts several photographs illustrating the decrease in amountsof Crp, Fur, RpoS and PhoP proteins as a consequence of growth of χ9021(ΔP_(crp527)), χ8848 (ΔP_(fur33)), χ8956 (ΔP_(rpoS183)) and χ8918(ΔP_(phoPQ107)) in the absence of arabinose. The same bacterial strainsas used for the results shown in FIG. 47, were grown in nutrient brothwith 0.2 percent arabinose and at the commencement of sampling tomeasure the amounts of proteins, the cultures were diluted 1:4 intoprewarmed nutrient broth lacking arabinose. Rabbit antibodies raisedagainst His-tagged Crp, Fur and PhoP were used for western blotanalyses. Mouse monoclonal antibody against RpoS was purchased fromNeoclone. Synthesis of the Crp, Fur and PhoP proteins continues untilafter the third 1:4 dilution whereas the amount of the RpoS proteindecreases conciderably after the first 1:4 dilution.

FIG. 30 depicts a photograph of the LPS profiles of vaccine strains insilver-stained SDS-PAGE. Bacteria were lysed with SDS, treated withproteinase K, and analyzed by SDS-PAGE followed by LPS-specific silverstaining. Lane 1, □ χ9088(pYA3634) (grown in nutrient broth withoutmannose); Lane 2, χ9558(pYA3634) (grown in nutrient broth withoutmannose); Lane 3, χ9088(pYA3634) (grown in nutrient broth with 0.5%mannose); Lane 4, χ9558(pYA3634) (grown in nutrient broth with 0.5%mannose); Lane 5, □ χ9088(pYA3634) (grown in LB broth); Lane 6,χ9558(pYA3634) (grown in LB broth).

FIG. 31 depicts an illustration showing different regulated delayedattenuation constructs in vaccine strains. To create the Δpmi-2426mutation, 1176 bp of the pmi nucleic acid sequence was deleted (from ATGto TAG). To create the ΔP_(crp527)::TT araC P_(BAD) crp mutation, 95 bpof the crp promoter region (−15 to −109) was deleted and 1335 bp ofP_(BAD) araC TT was inserted. To create the ΔP_(fur33)::TT araC P_(BAD)fur mutation, 239 bp of the fur promoter region (−15 to −253; includingFur consenus, Crp binding, and OxyR binding sites) was deleted and 1335bp of P_(BAD) araC TT was inserted.

FIG. 32 depicts a photograph showing western blot data of the synthesisof PspA Rx1 by different S. typhimurium mutants. Cell lysates of S.typhimurium mutants containing PspA Rx1 were subjected to SDS-PAGE, andthe proteins were transferred to nitrocellulose, which was subsequentlyprobed with a polyclonal antibody specific for PspA Lanes: 1, molecularmass markers (positions are indicated in kilodaltons); 2,χ8133(pYA3493); 3, □ χ8133(pYA3634); 4, □ χ9088(pYA3493); 5, □χ9088(pYA3634); 6, □ χ9558(pYA3493); 7, □ χ9558(pYA3634). Due to thepresence of arabinose in LB broth, the PspA synthesis of χ9558(pYA3634)has been suppressed partly.

FIG. 33A, FIG. 33B and FIG. 33C depict a series of graphs showing serumIgG responses to rPspA (FIG. 33A) and to S. typhimurium LPS (FIG. 33B)and SOMPs (FIG. 33C) measured by ELISA. The data represent IgG antibodylevels in mice orally immunized with χ8133(pYA3493) (vector control),χ8133(pYA3634) (expressing rPspA), χ9088(pYA3493) (vector control),χ9088(pYA3634) (expressing rPspA), χ9558(pYA3493) (vector control) andχ9558(pYA3634) (expressing rPspA) at the indicated weeks afterimmunization. p<0.05 for anti-rPspA serum IgG antibody levels ofχ9558(pYA3634) and χ9088(pYA3634) immunized mice with that of theχ8133(pYA3634) immunized mice at week 8. p<0.01 for the anti-rPspA serumIgG antibody levels of χ9558(pYA3634) immunized mice compared toχ8133(pYA3634) immunized mice at week 10 and 12. p<0.05 for theanti-rPspA serum IgG levels of χ9558(pYA3634) immunized mice compared toχ9088(pYA3634) immunized mice at week 8, 10 and 12.

FIG. 34A, FIG. 34B and FIG. 34C depict a series of graphs showing serumIgG2a and IgG1 responses to rPspA measure by ELISA. The data representIgG2a and IgG1 subclass antibody levels to rPspA in sera of BALB/c miceorally immunized with the indicated strains at various times afterimmunization. The ratios of IgG1: IgG2a at 12 weeks are 1:8.3 forχ8133(pYA3634) immunized mice (FIG. 34A), 1:9.4 for χ9088(pYA3634)immunized mice (FIG. 34B) and 1:1.5 for χ9558(pYA3634) immunized mice(FIG. 34C).

FIG. 35A and FIG. 35B depict a series of graphs showing antigen-specificstimulation of IFN-γ (FIG. 35A) or IL-4 (FIG. 35B) production.Splenectomies were performed on euthanized BALB/c mice at 8 weeksfollowing immunization. BSG controls were also included. Splenocyteswere harvested from three mice per group. ELISPOT analyses wereperformed as described in Materials and Methods. The results arepresented as ELISPOTS per million splenocytes minus any backgroundELISPOTS from unpulsed mock controls. One-way ANOVA and LSD methods wereadopted to compare the secretion levels of IL-4 or IFN-γ betweendifferent groups. p<0.001 when compare χ9558(pYA3634) and χ9088(pYA3634)with χ8133(pYA3634) for both secretion levels of IL-4 and IFN-γ. p<0.01when compare χ9558(pYA3634) with χ9088(pYA3634) for the secretion levelsof IFN-γ.

FIG. 36A and FIG. 36B depict photomicrographs of conventional lightmicroscopy of H&E-stained lung tissue samples of WU2 challenged mice(10χ40). (FIG. 36A) S. pneumoniae caused focal consolidation withextensive mononuclear and polymorphonuclear infiltration and loss ofalveolar structure in mice that succumbed to the infection. (FIG. 36B)Lungs from survivors appeared normal without extensive cellularinfiltration.

FIG. 37A and FIG. 37B depict the systhesis of Asd protein from the asdgene with ATG or GTG start codon and muramic acid-less death assay.(FIG. 37A) The western blot was performed with cell lysates of S.typhimurium strain χ8276 (ΔasdA16) and its derivatives cultured in LBbroth with 0.2% arabinose. DAP was included in the medium for strainχ8276. Asd protein was detected using rabbit anti-Asd serum. The 39 kDaAsd protein is indicated by an arrow. (FIG. 37B) Growth of Salmonellastrain χ8645 (ΔP_(murA7)::araC P_(BAD) murA) with and without arabinosein the indicated media.

FIG. 38A and FIG. 38B depict an illustration and a series of photographsshowing the defined deletion mutations of strain χ8937 and nutritionalrequirements. (FIG. 38A) The defined deletion chromosomal mutations inwild-type Salmonella UK-1 and in strain χ8937. P: promoter, TT:transcriptional terminator. (FIG. 38B) The growth of host strain χ8937alone or strain χ8937 harboring pYA3681 on LB agar plates with orwithout supplementations.

FIG. 39 depicts a diagram illustrating the regulatory interactions inthe programmed lysis system.

FIG. 40A, FIG. 40B and FIG. 40C depict a series of graphs illustratingthe in vitro and in vivo lysis of the programmed lysis system in theabsence of arabinose. (FIG. 40A) The growth curves of strainχ8937(pYA3681) with arabinose-regulated asdA and murA expression in LBbroth with or without the addition of 0.02% arabinose. (FIG. 40B) Theratio of released β-galactosidase versus total β-galactosidase whenstrain χ9380(pYA3681) with arabinose-regulated asdA and murA expressionand constitutive lacZ expression was grown in LB broth with or without0.02% arabinose; the wild-type strain χ9379 modified to express lacZacts as a non-lysis system control. (FIG. 40C) Colonization of mice withS. typhimurium χ8937(pYA3681) following P.O. inoculation with 10⁹ CFUbacteria. The limits of detection for this assay were 10 CFU/PP or g oftissue.

FIG. 41A, FIG. 41B and FIG. 41C depict the synthesis of PspA Rx1 andarabinose-dependent growth of χ8937(pYA3685). (FIG. 41A) Map of plasmidpYA3685. Plasmid sequences include the trpA, rrfG and 5S ribosomal RNAtranscriptional terminators, the P_(BAD), P_(trc) and P22 P_(R)promoters, the araC gene and start codon-modified murA and asdA genes,and the bla-pspA fusion protein. (FIG. 41B) The synthesis of rPspA Rx1in the programmed lysis S. typhimurium strain χ8937(pYA3685) grown in LBbroth with 0.02% arabinose at 37° C. Aliquots of mid-log phase cultureswere subjected to SDS-PAGE or immunoblot analysis. The immunoblot wasprobed with anti-PspA antibody. PspA proteins are indicated by arrows.Lanes 1 and 2, protein from χ8937(pYA3685) and χ8937(pYA3681),respectively. (FIG. 41C) Growth curves of PspA-producing strainχ8937(pYA3685) in LB broth with or without the addition of 0.02%arabinose.

FIG. 42A and FIG. 42B depict a series of graphs showing Immune responsesin mice after oral immunization with χ8937(pYA3685) (rPspA Rx1) andχ8937(pYA3681) (vector control) as determined by ELISA. (FIG. 42A) IgGantibody against S. typhimurium SOMPs and rPspA Rx1 in a 1:1280 dilutionof serum. (FIG. 42B) Anti-SOMP and -rPspA Rx1 IgA antibody levels in a1:10 dilution of vaginal secretions.

FIG. 43 depicts a graph showing IgG isotype analyses. Serum IgG2a andIgG1 responses to SOMPs and rPspA. IgG2a (gray bars) and IgG1 (darkbars). Serum was diluted 1:400.

FIG. 44A, FIG. 44B and FIG. 44C depict a series of graphs showing thesensitivity of (FIG. 44A) χ9633(pYA4088), (FIG. 44B) χ9639(pYA4088) and(FIG. 44C) χ9640(pYA4088) RASV-Sp strains to low pH.

FIG. 45 depicts a graph showing the stability of RASV-Sp vaccine inEnsure nutrition shakes at 37° C.

FIG. 46 depicts a graph showing the stability of RASV-Sp strains in PBSat room temperature.

FIG. 47A, FIG. 47B and FIG. 47C depict a series of graphs showing thecolonization of the S. typhi strains in (FIG. 47A) instestine, (FIG.47B) spleen, and (FIG. 47C) liver of newborn mice.

FIG. 48A and FIG. 48B depict a series of graphs showing the (FIG. 48A)weights of guinea pigs administered sterile and cell-free PBS wash, and(FIG. 48B) weights of mice administered sterile and cell-free PBS wash.

FIG. 49A, FIG. 49B and FIG. 49C depict a schematic of PspA expressionplasmids (FIG. 49A) pYA4088 and (FIG. 49B) pYA3634 with empty controlvector (FIG. 49C) pYA3493.

FIG. 50A and FIG. 50B depict a series of graphs showing the total serumIgG from mice orally vaccinated with χ8133(pYA3634), χ9088(pYA3634) andχ9558(pYA3634) to (FIG. 50A) PspA and to (FIG. 50B) S. typhimurium LPS.

FIG. 51 depicts a graph showing immunization with χ9558(pYA3634)protects mice against challenge with virulent S. pneumoniae strain WU2.

FIG. 52A, FIG. 52B and FIG. 52C depict a series of graphs showing (FIG.52A) the total IgG antibody response to PspA, (FIG. 52B) the total IgGantibody response to S. typhi LPS, and (FIG. 52C) the total antibodyresponse to S. typhi outer membrane proteins.

FIG. 53A, FIG. 53B and FIG. 53C depict a series of graphs showing thesurvival of (FIG. 53A) S. typhi ISP1820 derivatives, (FIG. 53B) Ty2RpoS⁻ derivatives, and (FIG. 53C) Ty2 RpoS⁺ derivatives in active (A)and heat-inactivated (HI) whole human blood including χ8110 and Ty21a ascontrols.

FIG. 54 depicts a graph showing the resistance of RASV-Sp strainscompared to wild-type S. typhi strains to guinea pig complement.

FIG. 55A, FIG. 55B and FIG. 55C depict a series of graphs showing thesurvival of (FIG. 55A) S. typhi ISP1820 derivatives, (FIG. 55B) Ty2RpoS⁻ derivatives, and (FIG. 55C) Ty2 RpoS⁺ derivatives in peripheralblood mononuclear cells.

FIG. 56 depicts the survival of S. typhi in human stool.

FIG. 57A, FIG. 57B and FIG. 57C depict the survival of RASV-Sp strainsand wild-type S. typhi in (FIG. 57A) chlorinated water, (FIG. 57B)untreated canal water, and (FIG. 57C) raw sewage.

FIG. 58A, FIG. 58B, FIG. 58C and FIG. 58D depict the serum IgG responsesto rPspA (FIG. 58A), to S. typhi LPS (FIG. 58B), to OMPs (FIG. 58C) andsIgA (FIG. 58D) in immunized mice. Serum IgG responses against rPspA(FIG. 58A) S. typhi LPS (FIG. 58B), and SOMPS (FIG. 58C) and mucosal IgAresponses to rPspA (FIG. 58D) were measured by ELISA using pooled serafrom BALB/c mice intranasally immunized with the indicated strainscarrying either plasmid pYA3493 (negative control) or pYA4088 (PspA).Error bars represent variation between triplicate wells. Mice wereboosted at week 6. Statistical significance was determined at week 8. *,P<0.05; **, P<0.01 for χ9633(pYA4088), χ9639(pYA4088) and χ9640(pYA4088)were compared each other.

FIG. 59 depicts a schematic of the phase I safety and tolerabilityclinical study design.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides, in some embodiments, a recombinantbacterium capable of regulated expression of at least one nucleic acidsequence encoding an antigen of interest. In other embodiments, theinvention provides a recombinant bacterium capable of regulatedattenuation. In exemplary embodiments, the invention provides arecombinant bacterium capable of both regulated expression of at leastone nucleic acid sequence encoding an antigen of interest and regulatedattenuation.

In each of the embodiments herein, the recombinant bacterium typicallybelongs to the Enterobaceteriaceae. The Enterobacteria family comprisesspecies from the following genera: Alterococcus, Aquamonas, Aranicola,Arsenophonus, Brenneria, Budvicia, Buttiauxella, CandidatusPhlomobacter, Cedeceae, Citrobacter, Edwardsiella, Enterobacter,Erwinia, Escherichia, Ewingella, Hafnia, Klebsiella, Kluyvera,Leclercia, Leminorella, Moellerella, Morganella, Obesumbacterium,Pantoea, Pectobacterium, Photorhabdus, Plesiomonas, Pragia, Proteus,Providencia, Rahnella, Raoultella, Salmonella, Samsonia, Serratia,Shigella, Sodalis, Tatumella, Trabulsiella, Wigglesworthia, Xenorhbdus,Yersinia, Yokenella. In certain embodiments, the recombinant bacteriumis typically a pathogenic species of the Enterobaceteriaceae. Due totheir clinical significance, Escherichia coli, Shigella, Edwardsiella,Salmonella, Citrobacter, Klebsiella, Enterobacter, Serratia, Proteus,Morganella, Providencia and Yersinia are considered to be particularlyuseful. In other embodiments, the recombinant bacterium may be a speciesor strain commonly used for a vaccine.

Some embodiments of the instant invention comprise a species orsubspecies of the Salmonella genera. For instance, the recombinantbacterium may be a Salmonella enterica serovar. In an exemplaryembodiment, a bacterium of the invention may be derived from S.typhimurium, S. typhi, S. paratyphi, S. gallinarum, S. enteritidis, S.choleraesius, S. arizona, or S. dublin.

A recombinant bacterium of the invention derived from Salmonella may beparticularly suited to use as a vaccine. Infection of a host with aSalmonella strain typically leads to colonization of the gut-associatedlymphoid tissue (GALT) or Peyer's patches, which leads to the inductionof a generalized mucosal immune response to the recombinant bacterium.Further penetration of the bacterium into the mesenteric lymph nodes,liver and spleen may augment the induction of systemic and cellularimmune responses directed against the bacterium. Thus the use ofrecombinant Salmonella for oral immunization stimulates all threebranches of the immune system, which is particularly important forimmunizing against infectious disease agents that colonize on and/orinvade through mucosal surfaces.

In an alternative embodiment, a bacterium of the invention may be abacterium included in Table 1 below.

TABLE 1 Strain Genotype or relevant characteristics Escherichia coliχ289 F⁻ λ⁻ glnV42 T3^(r) χ6097 F⁻ araD139 Δ(proAB-lac) λ⁻ ϕ80dlacZΔM15rpsL ΔasdA4 Δ(zhf- 2::Tn10) thi-1 χ6212 F⁻ Δ(argF-lacZYA)-U169 glnV44 λ⁻deoR ϕ80dlacZΔM15 gyrA96 recA1 relA1 endA1 ΔasdA4 Δ(zhf-2::Tn10) thi-1hsdR17 χ7213 thr-1 leuB6 fhuA21 lacY1 glnV44 recA1 ΔasdA4 Δ(zhf-2::Tn10)thi-1 RP4-2-Tc::Mu [λpir]; Km^(r) χ7232 endA1 thr-1 hsdR17 (r_(K) ⁻,m_(K) ⁺) supE44 gyrA recA1 ΔrelA1 Δ(argF- lacZYA)-U169 [λpir] deoRΦ80dlacZΔM15 χ7370 F⁻ araD139 Δ(ara-leu)-7697 ΔlacX74 Δlon-4 galK deoRΔcsgA4 mcrA galU 

 80dlacZΔM15 ΔfliC38 Δ(wcaL-wza)-19 recA1 endA1 nupG rpsl Δ(fimA-H)Δ(mcrBC-hsdRMS-mrr) χ7385 F⁻ araD139 Δ(ara-leu)-7697 Δ(lacAYZOPI)-X74Δlon-4 ΔompT0523::TT araC P_(BAD) T7 pol TT galK deoR ΔcsgA4 mcrA galUϕ80dlacZΔM15 ΔfliC38 Δ(wcaL-wza)-19 recA1 endA1 nupG rpsL Δ(fimA-H)Δ(mcrBC-hsdRMS-mrr) ΔasdA99 BL21 (DE3) F⁻ ompT hsdS_(B)(r_(B) ⁻ _(,)m_(B) ⁻) gal dcm (DE3) Top 10 F⁻ mcrA Δ(mrr-hsdRMS-mcrBC) ϕ80dlacZΔM15ΔlacX74 recA1 araD139 Δ(ara-leu)7697 galU galK rpsL (Str^(r)) endA1 nupGXL1-blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proABlacI^(q)ZΔM15 Tn10 (Tet^(r))]. Salmonella enterica Typhimurium UK-1χ3761 UK-1 wild type χ8060 ΔpabA1516 χ8133 Δcya-27 Δcrp-27 ΔasdA16 χ8276ΔasdA16 χ8289 ΔasdA19::TTaraC P_(BAD) c2 χ8442 ΔpabA1516 ΔpabB232 χ8477ΔaraE25 χ8645 ΔP_(murA7)::TT araC P_(BAD) murA χ8767 ΔaraBAD23 χ8831Δ(gmd-fcl)-26 χ8844 ΔendA2311 χ8848 ΔP_(fur33)::TT araC P_(BAD) furχ8854 ΔendA2311 ΔasdA19::TT araC P_(BAD) c2 ΔP_(murA7)::TT araC P_(BAD)murA ΔaraE25 ΔaraBAD1923 χ8882 ΔrelA1123 χ8914 ΔpabA1516 ΔpabB232ΔasdA16 χ8918 ΔP_(phoPQ107)::TT araC P_(BAD) phoPQ χ8937 ΔasdA19::araCP_(BAD) c2 ΔP_(murA7)::araC P_(BAD) murA Δ(gmd-fcl)-26 ΔrelA1123ΔendA2311 χ8956 ΔP_(rpoS183)::TT araC P_(BAD) rpoS χ8960 ΔasdA18::TTaraC P_(BAD) c2 χ8989 ΔendA19::araC P_(BAD) lacI TT χ8990 ΔrelA196::araCP_(BAD) lacI TT χ9000 ΔP_(fur33)::TT araC P_(BAD) fur ΔP_(phoPQ107)::TTaraC P_(BAD) phoPQ χ9021 ΔP_(crp527)::TT araC P_(BAD) crp χ9064ΔP_(rpoS183)::TT araC P_(BAD) rpoS ΔP_(crp527)::TT araC P_(BAD) crpχ9080 ΔrelA197::araC P_(BAD) lacI TT χ9088 Δpmi-2426 Δ(gmd-fcl)-26ΔP_(fur33)::TT araC _(BAD) fur ΔasdA33 χ9095 ΔpabA1516 ΔpabB232 ΔasdA16ΔrelA196::araC P_(BAD) lacI TT ΔaraBAD23 χ9097 ΔpabA1516 ΔpabB232ΔasdA16 ΔaraBAD23 χ9101 ΔpabA1516 ΔpabB232 ΔasdA16 ΔrelA197::araCP_(BAD) lacI TT ΔaraBAD23 χ9107 ΔP_(fur33)::TT araC P_(BAD) furΔP_(crp527)::TT araC P_(BAD) crp χ9108 ΔP_(phoPQ107)::TT araC P_(BAD)phoPQ ΔP_(crp527)::TT araC P_(BAD) crp χ9109 ΔP_(fur33)::TT araC P_(BAD)fur ΔP_(phoPQ107)::TT araC P_(BAD) phoPQ ΔP_(crp527)::TT araC P_(BAD)crp χ9225 ΔendA21::araC P_(BAD) lacI TT χ9226 ΔrelA198::araC P_(BAD)lacI TT χ9241 ΔpabA1516 ΔpabB232 ΔasdA16 ΔaraBAD23 ΔrelA198::araCP_(BAD) lacI TT χ9269 ΔP_(fur81)::TT araC P_(BAD) fur χ9273ΔP_(fur77)::TT araC P_(BAD) fur χ9275 ΔasdA21::TT araC P_(BAD) c2 χ9302ΔasdA20::TT araC P_(BAD) c2 χ9339 ΔsifA26 ΔasdA18::TT araC P_(BAD) c2ΔP_(crp527)::TT araC P_(BAD) crp ΔaraBAD23 χ9340 Δalr-3 ΔdadB4ΔP_(phoPQ107) ::TT araC P_(BAD) phoPQ ΔrecJ1516 ΔrecF126 ΔasdA18::TTaraC P_(BAD) c2 χ9362 Δpmi-2426 Δ (gmd-fcl)-26 ΔP_(phoPQ107)::TT araCP_(BAD) phoPQ ΔP_(crp527)::TT araC P_(BAD) crp ΔasdA18::TT araC P_(BAD)c2 ΔaraE25 ΔaraBAD23 ΔrelA198::araC P_(BAD) lacI TT χ9371ΔP_(phoPQ173)::TT araC P_(BAD) phoPQ χ9372 ΔP_(phoPQ177)::TT araCP_(BAD) phoPQ χ9373 Δpmi-2426 Δ(gmd-fcl)-26 ΔP_(fur81)::TT araC P_(BAD)fur ΔP_(crp527)::TT araC P_(BAD) crp ΔasdA21::TT araC P_(BAD) c2 ΔaraE25ΔaraBAD23 ΔrelA198::araC P_(BAD) lacI TT χ9379 χ3761 ΔatrB13::MudJ χ9380χ9379 ΔatrB13::MudJ χ9382 ΔP_(phoPQ173)::TT araC P_(BAD) phoPQ ΔaraBAD23χ9383 ΔP_(phoPQ177)::TT araC P_(BAD) phoPQ ΔaraBAD23 χ9402 Δpmi-2426Δ(gmd-fcl)-26 ΔP_(fur81)::TT araC P_(BAD) fur ΔP_(crp527)::TT araCP_(BAD) crp ΔasdA21::TT araC P_(BAD) c2 ΔaraE25 ΔaraBAD23 ΔrelA198::araCP_(BAD) lacI TT ΔsopB1925 χ9412 Δpmi-2426 Δ(gmd-fcl)-26 ΔP_(fur81)::TTaraC P_(BAD) fur ΔP_(crp527)::TT araC P_(BAD) crp ΔasdA21::TT araCP_(BAD) c2 ΔaraE25 ΔaraBAD23 ΔrelA198::araC P_(BAD) lacI TTΔP_(murA7)::TT araC P_(BAD) murA χ9413 Δpmi-2426 Δ(gmd-fcl)-26ΔP_(phoPQ107)::TT araC P_(BAD) phoPQ ΔP_(crp527)::TT araC P_(BAD) crp ΔasdA18::TT araC P_(BAD) c2 Δ araE25 Δ araBAD23 Δ relA198::araC P_(BAD)lacI TT Δ P_(murA7)::TT araC P_(BAD) murA χ9442 Δ P_(murA12)::TT araCP_(BAD) murA χ9443 Δ (araC P_(BAD))-5::P22 P_(R) araBAD χ9444 ΔasdA34::TT χ9477 Δ asdA27::TT araC P_(BAD) c2 χ9509 Δ relA198::araCP_(BAD) lacI TT Δ araBAD23 χ9521 Δ P_(murA12)::TT araC P_(BAD) murA Δ(araC P_(BAD))-5::P22 P_(R) araBAD χ9527 Δ P_(fur77)::TT araC P_(BAD)fur Δ (araC P_(BAD))-5::P22 P_(R) araBAD χ9533 Δ P_(crp527)::TT araCP_(BAD) crp Δ (araC P_(BAD))-5::P22 P_(R) araBAD χ9541 ΔP_(phoPQ176)::TT araC P_(BAD) phoPQ χ9542 Δ P_(phoPQ175)::TT araCP_(BAD) phoPQ χ9543 Δ P_(phoPQ174)::TT araC P_(BAD) phoPQ χ9548 ΔP_(phoPQ175)::TT araC P_(BAD) phoPQ Δ araBAD23 χ9549 Δ P_(phoPQ174)::TTaraC P_(BAD) phoPQ Δ araBAD23 χ9550 Δ P_(fur77)::TT araC P_(BAD) fur ΔP_(crp527)::TT araC P_(BAD) crp χ9551 Δ asdA34::TT Δ P_(phoPQ107)::TTaraC P_(BAD) phoPQ χ9552 Δ asdA34::TT Δ P_(phoPQ173)::TT araC P_(BAD)phoPQ χ9553 Δ asdA34::TT Δ P_(phoPQ177)::TT araC P_(BAD) phoPQ □□χ9558Δpmi-2426 Δ(gmd-fcl)-26 ΔP_(fur81)::TT araC P_(BAD) fur ΔP_(crp527)::TTaraC P_(BAD) crp ΔasdA27::TT araC P_(BAD) c2 ΔaraE25 ΔaraBAD23ΔrelA198::araC P_(BAD) lacI TT ΔsopB1925 ΔagfBAC811 χ9569 Δ endA20::araCP_(BAD) lacI TT Salmonella enterica Typhi ISP1820 χ9421 Δ P_(crp527)::TTaraC P_(BAD) crp Δ P_(fur81)::TT araC P_(BAD) fur Δ P_(phoPQ107)::TTaraC P_(BAD) phoPQ Δ pmi-2426 Δ (gmd-fcl)-26 Δ sopB1925 Δ relA198::araCP_(BAD) lacI TT Δ araE25 ΔaraBAD23 Δ tviABCDE10 Δ agfBAC811 χ9633ΔP_(crp527)::TT araC P_(BAD) crp ΔP_(fur81)::TT araC P_(BAD) furΔpmi-2426 Δ(gmd-fcl)-26 ΔsopB1925 ΔrelA198::araC P_(BAD) lacI TT ΔaraE25ΔaraBAD23 ΔtviABCDE10 ΔagfBAC811 PhoP⁺ ΔasdA33 Salmonella enterica TyphiTy2 χ9205 Δ P_(crp527)::TT araC P_(BAD) crp Δ P_(fur33)::TT araC P_(BAD)fur RpoS⁻ χ9114 Δ P_(phoPQ107)::TT araC P_(BAD) phoPQ ΔP_(crp527)::TTaraC P_(BAD) crp RpoS⁻ χ9213 Δ P_(crp527)::TT araC P_(BAD)crp Δ P_(fur33)::TT araC P_(BAD) fur Δ P_(phoPQ107)::TT araC P_(BAD)phoPQ RpoS⁻ χ9369 Δ P_(crp527)::TT araC P_(BAD) crp Δ P_(fur33)::TT araCP_(BAD) fur Δ P_(phoPQ107)::TT araC P_(BAD) phoPQ Δ pmi-2426 Δgmd-fcl-26 Δ relA198::araC P_(BAD) lacI TT RpoS⁻ χ9639 ΔP_(crp527)::TTaraC P_(BAD) crp ΔP_(fur81)::TT araC P_(BAD) fur Δpmi-2426 Δ(gmd-fcl)-26ΔsopB1925 ΔrelA198::araC P_(BAD) lacI TT ΔaraE25 ΔtviABCDE10 ΔagfBAC811PhoP⁺ ΔasdA33 RpoS⁻ χ9640 ΔP_(crp527)::TT araC P_(BAD) crpΔP_(fur81)::TT araC P_(BAD) fur Δpmi-2426 Δ(gmd-fcl)-26 ΔsopB1925ΔrelA198::araC P_(BAD) lacI TT ΔaraE25 ΔtviABCDE10 ΔagfBAC811 PhoP⁺RpoS⁺ ΔasdA33 Salmonella enterica Paratyphi A χ9515 Δ P_(crp527)::TTaraC P_(BAD) crp Δ P_(fur81)::TT araC P_(BAD) fur Δ P_(phoPQ107)::TTaraC P_(BAD) phoPQ Δ pmi-2426 Δ (gmd-fcl)-26 Δ sopB1925 Δ agfBAC811 ΔrelA198::araC P_(BAD) lacI TT χ9608 Δ P_(crp527)::TT araC P_(BAD) crp ΔP_(fur81)::TT araC P_(BAD) fur Δ P_(phoPQ107)::TT araC P_(BAD) phoPQ Δpmi-2426 Δ (gmd-fcl)-26 ΔagfBAC811 Δ relA198::araC P_(BAD) lacI TT ΔsopB1925 ΔaraE25 ΔaraBAD23 ΔasdA33 χ9651 ΔP_(crp527)::TT araC P_(BAD)crp ΔP_(fur81)::TT araC P_(BAD) fur ΔP_(phoPQ107)::TT araC P_(BAD) phoPQΔpmi-2426 Δ(gmd-fcl)-26 ΔagfBAC811 ΔrelA198::araC P_(BAD) lacI TTΔsopB1925 ΔaraE25 Δ(araC P_(BAD))-5::P22 P_(R) araBAD χ9763 ΔP_(crp527)::TT araC P_(BAD) crp Δ P_(fur81)::TT araC P_(BAD) fur Δpmi-2426 Δ (gmd-fcl)-26 Δ agfBAC811 Δ relA198::araC P_(BAD) lacI TT ΔsopB1925 ΔaraE25 ΔaraBAD23 PhoP⁺ χ9857 Δ P_(crp527)::TT araC P_(BAD) crpΔ P_(fur81)::TT araC P_(BAD) fur Δ pmi-2426 Δ (gmd-fcl)-26 Δ agfBAC811 ΔrelA198::araC P_(BAD) lacI TT Δ sopB1925 ΔaraE25 ΔaraBAD23 PhoP⁺ ΔasdA33Streptococcus pneumoniae Rx1 PspA Clade 2, Capsule Type Rough WU2 PspAClade 2, Capsule Type 3 D39 PspA Clade 2, Capsule Type 2 Δ = deletion P= promoter p = plasmid TT = transcription terminatorI. Regulated Expression

The present invention encompasses a recombinant bacterium capable ofregulated expression of at least one nucleic acid sequence encoding anantigen of interest. Generally speaking, the bacterium comprises achromosomally integrated nucleic acid sequence encoding a repressor anda vector. Each is discussed in more detail below.

(a) Chromosomally Integrated Nucleic Acid Sequence Encoding a Repressor

A recombinant bacterium of the invention that is capable of theregulated expression of at least one nucleic acid sequence encoding anantigen comprises, in part, at least one chromosomally integratednucleic acid sequence encoding a repressor. Typically, the nucleic acidsequence encoding a repressor is operably linked to a regulatablepromoter. The nucleic acid sequence encoding a repressor and/or thepromoter may be modified from the wild-type nucleic acid sequence so asto optimize the expression level of the nucleic acid sequence encodingthe repressor.

Methods of chromosomally integrating a nucleic acid sequence encoding arepressor operably-linked to a regulatable promoter are known in the artand detailed in the examples. Generally speaking, the nucleic acidsequence encoding a repressor should not be integrated into a locus thatdisrupts colonization of the host by the recombinant bacterium, orattenuates the bacterium. In one embodiment, the nucleic acid sequenceencoding a repressor may be integrated into the relA nucleic acidsequence. In another embodiment, the nucleic acid sequence encoding arepressor may be integrated into the endA nucleic acid sequence.

In some embodiments, at least one nucleic acid sequence encoding arepressor is chromosomally integrated. In other embodiments, at leasttwo, or at least three nucleic acid sequences encoding repressors may bechromosomally integrated into the recombinant bacterium. If there ismore than one nucleic acid sequence encoding a repressor, each nucleicacid sequence encoding a repressor may be operably linked to aregulatable promoter, such that each promoter is regulated by the samecompound or condition. Alternatively, each nucleic acid sequenceencoding a repressor may be operably linked to a regulatable promoter,each of which is regulated by a different compound or condition.

i. Repressor

As used herein, “repressor” refers to a biomolecule that repressestranscription from one or more promoters. Generally speaking, a suitablerepressor of the invention is synthesized in high enough quantitiesduring the in vitro growth of the bacterial strain to repress thetranscription of the nucleic acid encoding an antigen of interest on thevector, as detailed below, and not impede the in vitro growth of thestrain. Additionally, a suitable repressor will generally besubstantially stable, i.e. not subject to proteolytic breakdown.Furthermore, a suitable repressor will be diluted by about half at everycell division after expression of the repressor ceases, such as in anon-permissive environment (e.g. an animal or human host).

The choice of a repressor depends, in part, on the species of therecombinant bacterium used. For instance, the repressor is usually notderived from the same species of bacteria as the recombinant bacterium.For instance, the repressor may be derived from E. coli if therecombinant bacterium is from the genus Salmonella. Alternatively, therepressor may be from a bacteriophage.

Suitable repressors are known in the art, and may include, for instance,Lacl of E. coli, C2 encoded by bacteriophage P22, or C1 encoded bybacteriophage A. Other suitable repressors may be repressors known toregulate the expression of a regulatable nucleic acid sequence, such asnucleic acid sequences involved in the uptake and utilization of sugars.In one embodiment, the repressor is Lacl. In another embodiment, therepressor is C2. In yet another embodiment, the repressor is C1.

ii. Regulatable Promoter

The chromosomally integrated nucleic acid sequence encoding a repressoris operably linked to a regulatable promoter. The term “promoter”, asused herein, may mean a synthetic or naturally-derived molecule that iscapable of conferring, activating or enhancing expression of a nucleicacid. A promoter may comprise one or more specific transcriptionalregulatory sequences to further enhance expression and/or to alter thespatial expression and/or temporal expression of a nucleic acid. Theterm “operably linked,” as used herein, means that expression of anucleic acid is under the control of a promoter with which it isspatially connected. A promoter may be positioned 5′ (upstream) of thenucleic acid under its control. The distance between the promoter and anucleic acid to be expressed may be approximately the same as thedistance between that promoter and the native nucleic acid sequence itcontrols. As is known in the art, variation in this distance may beaccommodated without loss of promoter function.

The regulated promoter used herein generally allows transcription of thenucleic acid sequence encoding a repressor while in a permissiveenvironment (i.e. in vitro growth), but ceases transcription of thenucleic acid sequence encoding a repressor while in a non-permissiveenvironment (i.e. during growth of the bacterium in an animal or humanhost). For instance, the promoter may be sensitive to a physical orchemical difference between the permissive and non-permissiveenvironment. Suitable examples of such regulatable promoters are knownin the art.

In some embodiments, the promoter may be responsive to the level ofarabinose in the environment. Generally speaking, arabinose may bepresent during the in vitro growth of a bacterium, while typicallyabsent from host tissue. In one embodiment, the promoter is derived froman araC-P_(BAD) system. The araC-P_(BAD) system is a tightly regulatedexpression system which has been shown to work as a strong promoterinduced by the addition of low levels of arabinose (5). The araC-araBADpromoter is a bidirectional promoter controlling expression of thearaBAD nucleic acid sequences in one direction, and the araC nucleicacid sequence in the other direction. For convenience, the portion ofthe araC-araBAD promoter that mediates expression of the araBAD nucleicacid sequences, and which is controlled by the araC nucleic acidsequence product, is referred to herein as P_(BAD). For use as describedherein, a cassette with the araC nucleic acid sequence and thearaC-araBAD promoter may be used. This cassette is referred to herein asaraC-P_(BAD). The AraC protein is both a positive and negative regulatorof P_(BAD). In the presence of arabinose, the AraC protein is a positiveregulatory element that allows expression from P_(BAD). In the absenceof arabinose, the AraC protein represses expression from P_(BAD). Thiscan lead to a 1,200-fold difference in the level of expression fromP_(BAD).

Other enteric bacteria contain arabinose regulatory systems homologousto the araC araBAD system from E. coli. For example, there is homologyat the amino acid sequence level between the E. coli and the S.typhimurium AraC proteins, and less homology at the DNA level. However,there is high specificity in the activity of the AraC proteins. Forexample, the E. coli AraC protein activates only E. coli P_(BAD) (in thepresence of arabinose) and not S. typhimurium P_(BAD). Thus, anarabinose regulated promoter may be used in a recombinant bacterium thatpossesses a similar arabinose operon, without substantial interferencebetween the two, if the promoter and the operon are derived from twodifferent species of bacteria.

Generally speaking, the concentration of arabinose necessary to induceexpression is typically less than about 2%. In some embodiments, theconcentration is less than about 1.5%, 1%, 0.5%, 0.2%, 0.1%, or 0.05%.In other embodiments, the concentration is 0.05% or below, e.g. about0.04%, 0.03%, 0.02%, or 0.01%. In an exemplary embodiment, theconcentration is about 0.05%.

In other embodiments, the promoter may be responsive to the level ofmaltose in the environment. Generally speaking, maltose may be presentduring the in vitro growth of a bacterium, while typically absent fromhost tissue. The malT nucleic acid encodes MalT, a positive regulator offour maltose-responsive promoters (P_(PQ), P_(EFG), P_(KBM), and P_(S)).The combination of malT and a mal promoter creates a tightly regulatedexpression system that has been shown to work as a strong promoterinduced by the addition of maltose (6). Unlike the araC-P_(BAD) system,malT is expressed from a promoter (P_(T)) functionally unconnected tothe other mal promoters. P_(T) is not regulated by MalT. ThemalEFG-malKBM promoter is a bidirectional promoter controllingexpression of the malKBM nucleic acid sequences in one direction, andthe malEFG nucleic acid sequences in the other direction. Forconvenience, the portion of the malEFG-malKBM promoter that mediatesexpression of the malKBM nucleic acid sequence, and which is controlledby the malT nucleic acid sequence product, is referred to herein asP_(KBM), and the portion of the malEFG-malKBM promoter that mediatesexpression of the malEFG nucleic acid sequence, and that is controlledby the malT nucleic acid sequence product, is referred to herein asP_(EFG). Full induction of P_(KBM) requires the presence of the MalTbinding sites of P_(EFG). For use in the vectors and systems describedherein, a cassette with the malT nucleic acid sequence and one of themal promoters may be used. This cassette is referred to herein asmalT-P_(mal). In the presence of maltose, the MalT protein is a positiveregulatory element that allows expression from P_(mal).

In still other embodiments, the promoter may be sensitive to the levelof rhamnose in the environment. Analogous to the araC-P_(BAD) systemdescribed above, the rhaRS-P_(rhaB) activator-promoter system is tightlyregulated by rhamnose. Expression from the rhamnose promoter (P_(rha))is induced to high levels by the addition of rhamnose, which is commonin bacteria but rarely found in host tissues. The nucleic acid sequencesrhaBAD are organized in one operon that is controlled by the P_(rhaBAD)promoter. This promoter is regulated by two activators, RhaS and RhaR,and the corresponding nucleic acid sequences belong to one transcriptionunit that is located in the opposite direction of the rhaBAD nucleicacid sequences. If L-rhamnose is available, RhaR binds to the P_(rhaRS)promoter and activates the production of RhaR and RhaS. RhaS togetherwith L-rhamnose in turn binds to the P_(rhaBAD) and the P_(rhaT)promoter and activates the transcription of the structural nucleic acidsequences (7). Full induction of rhaBAD transcription also requiresbinding of the Crp-cAMP complex, which is a key regulator of cataboliterepression (7).

Although both L-arabinose and L-rhamnose act directly as inducers forexpression of regulons for their catabolism, important differences existin regard to the regulatory mechanisms. L-Arabinose acts as an inducerwith the activator AraC in the positive control of the arabinoseregulon. However, the L-rhamnose regulon is subject to a regulatorycascade; it is therefore subject to even tighter control than the araCP_(BAD) system. L-Rhamnose acts as an inducer with the activator RhaRfor synthesis of RhaS, which in turn acts as an activator in thepositive control of the rhamnose regulon. In the present invention,rhamnose may be used to interact with the RhaR protein and then the RhaSprotein may activate transcription of a nucleic acid sequenceoperably-linked to the P_(rhaBAD) promoter.

In still other embodiments, the promoter may be sensitive to the levelof xylose in the environment. The xyR-P_(xyA) system is anotherwell-established inducible activator-promoter system. Xylose inducesxylose-specific operons (xylE, xylFGHR, and xylAB) regulated by XylR andthe cyclic AMP-Crp system. The XylR protein serves as a positiveregulator by binding to two distinct regions of the xyl nucleic acidsequence promoters. As with the araC-P_(BAD) system described above, thexylR-P_(xylAB) and/or xylR-P_(xylFGH) regulatory systems may be used inthe present invention. In these embodiments, xylR P_(xylAB) xyloseinteracting with the XylR protein activates transcription of nucleicacid sequences operably-linked to either of the two P_(xyl) promoters.

The nucleic acid sequences of the promoters detailed herein are known inthe art, and methods of operably-linking them to a chromosomallyintegrated nucleic acid sequence encoding a repressor are known in theart and detailed in the examples.

iii. Modification to Optimize Expression

A nucleic acid sequence encoding a repressor and regulatable promoterdetailed above, for use in the present invention, may be modified so asto optimize the expression level of the nucleic acid sequence encodingthe repressor. The optimal level of expression of the nucleic acidsequence encoding the repressor may be estimated, or may be determinedby experimentation (see the Examples). Such a determination should takeinto consideration whether the repressor acts as a monomer, dimer,trimer, tetramer, or higher multiple, and should also take intoconsideration the copy number of the vector encoding the antigen ofinterest, as detailed below. In an exemplary embodiment, the level ofexpression is optimized so that the repressor is synthesized while inthe permissive environment (i.e. in vitro growth) at a level thatsubstantially inhibits the expression of the nucleic acid encoding anantigen of interest, and is substantially not synthesized in anon-permissive environment, thereby allowing expression of the nucleicacid encoding an antigen of interest.

As stated above, the level of expression may be optimized by modifyingthe nucleic acid sequence encoding the repressor and/or promoter. Asused herein, “modify” refers to an alteration of the nucleic acidsequence of the repressor and/or promoter that results in a change inthe level of transcription of the nucleic acid sequence encoding therepressor, or that results in a change in the level of synthesis of therepressor. For instance, in one embodiment, modify may refer to alteringthe start codon of the nucleic acid sequence encoding the repressor.Generally speaking, a GTG or TTG start codon, as opposed to an ATG startcodon, may decrease translation efficiency ten-fold. In anotherembodiment, modify may refer to altering the Shine-Dalgarno (SD)sequence of the nucleic acid sequence encoding the repressor. The SDsequence is a ribosomal binding site generally located 6-7 nucleotidesupstream of the start codon. The SD consensus sequence is AGGAGG, andvariations of the consensus sequence may alter translation efficiency.In yet another embodiment, modify may refer to altering the distancebetween the SD sequence and the start codon. In still anotherembodiment, modify may refer to altering the −35 sequence for RNApolymerase recognition. In a similar embodiment, modify may refer toaltering the −10 sequence for RNA polymerase binding. In an additionalembodiment, modify may refer to altering the number of nucleotidesbetween the −35 and −10 sequences. In an alternative embodiment, modifymay refer to optimizing the codons of the nucleic acid sequence encodingthe repressor to alter the level of translation of the mRNA encoding therepressor. For instance, non-A rich codons initially after the startcodon of the nucleic acid sequence encoding the repressor may notmaximize translation of the mRNA encoding the repressor. Similarly, thecodons of the nucleic acid sequence encoding the repressor may bealtered so as to mimic the codons from highly synthesized proteins of aparticular organism. In a further embodiment, modify may refer toaltering the GC content of the nucleic acid sequence encoding therepressor to change the level of translation of the mRNA encoding therepressor.

In some embodiments, more than one modification or type of modificationmay be performed to optimize the expression level of the nucleic acidsequence encoding the repressor. For instance, at least one, two, three,four, five, six, seven, eight, or nine modifications, or types ofmodifications, may be performed to optimize the expression level of thenucleic acid sequence encoding the repressor.

By way of non-limiting example, when the repressor is Lacl, then thenucleic acid sequence of Lacl and the promoter may be altered so as toincrease the level of Lacl synthesis. In one embodiment, the start codonof the Lacl repressor may be altered from GTG to ATG. In anotherembodiment, the SD sequence may be altered from AGGG to AGGA. In yetanother embodiment, the codons of lacl may be optimized according to thecodon usage for highly synthesized proteins of Salmonella. In a furtherembodiment, the start codon of lacl may be altered, the SD sequence maybe altered, and the codons of lacl may be optimized.

Methods of modifying the nucleic acid sequence encoding the repressorand/or the regulatable promoter are known in the art and detailed in theexamples.

iv. Transcription Termination Sequence

In some embodiments, the chromosomally integrated nucleic acid sequenceencoding the repressor further comprises a transcription terminationsequence. A transcription termination sequence may be included toprevent inappropriate expression of nucleic acid sequences adjacent tothe chromosomally integrated nucleic acid sequence encoding therepressor and regulatable promoter.

(b) Vector

A recombinant bacterium of the invention that is capable of theregulated expression of at least one nucleic acid sequence encoding anantigen comprises, in part, a vector. The vector comprises a nucleicacid sequence encoding at least one antigen of interest operably linkedto a promoter. The promoter is regulated by the chromosomally encodedrepressor, such that the expression of the nucleic acid sequenceencoding an antigen is repressed during in vitro growth of thebacterium, but the bacterium is capable of high level synthesis of theantigen in an animal or human host.

As used herein, “vecto0r” refers to an autonomously replicating nucleicacid unit. The present invention can be practiced with any known type ofvector, including viral, cosmid, phasmid, and plasmid vectors. The mostpreferred type of vector is a plasmid vector.

As is well known in the art, plasmids and other vectors may possess awide array of promoters, multiple cloning sequences, transcriptionterminators, etc., and vectors may be selected so as to control thelevel of expression of the nucleic acid sequence encoding an antigen bycontrolling the relative copy number of the vector. In some instances inwhich the vector might encode a surface localized adhesin as theantigen, or an antigen capable of stimulating T-cell immunity, it may bepreferable to use a vector with a low copy number such as at least two,three, four, five, six, seven, eight, nine, or ten copies per bacterialcell. A non-limiting example of a low copy number vector may be a vectorcomprising the pSC101 ori.

In other cases, an intermediate copy number vector might be optimal forinducing desired immune responses. For instance, an intermediate copynumber vector may have at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 copies per bacterial cell.A non-limiting example of an intermediate copy number vector may be avector comprising the p15A ori.

In still other cases, a high copy number vector might be optimal for theinduction of maximal antibody responses. A high copy number vector mayhave at least 31, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or100 copies per bacterial cell. In some embodiments, a high copy numbervector may have at least 100, 125, 150, 175, 200, 225, 250, 275, 300,325, 350, 375, or 400 copies per bacterial cell. Non-limiting examplesof high copy number vectors may include a vector comprising the pBR orior the pUC ori.

Additionally, vector copy number may be increased by selecting formutations that increase plasmid copy number. These mutations may occurin the bacterial chromosome but are more likely to occur in the plasmidvector.

Preferably, vectors used herein do not comprise antibiotic resistancemarkers to select for maintenance of the vector.

i. Antigen

As used herein, “antigen” refers to a biomolecule capable of elicitingan immune response in a host. In some embodiments, an antigen may be aprotein, or fragment of a protein, or a nucleic acid. In an exemplaryembodiment, the antigen elicits a protective immune response. As usedherein, “protective” means that the immune response contributes to thelessening of any symptoms associated with infection of a host with thepathogen the antigen was derived from or designed to elicit a responseagainst. For example, a protective antigen from a pathogen, such asMycobacterium, may induce an immune response that helps to amelioratesymptoms associated with Mycobacterium infection or reduce the morbidityand mortality associated with infection with the pathogen. The use ofthe term “protective” in this invention does not necessarily requirethat the host is completely protected from the effects of the pathogen.

Antigens may be from bacterial, viral, mycotic and parasitic pathogens,and may be designed to protect against bacterial, viral, mycotic, andparasitic infections, respectively. Alternatively, antigens may bederived from gametes, provided they are gamete specific, and may bedesigned to block fertilization. In another alternative, antigens may betumor antigens, and may be designed to decrease tumor growth. It isspecifically contemplated that antigens from organisms newly identifiedor newly associated with a disease or pathogenic condition, or new oremerging pathogens of animals or humans, including those now known oridentified in the future, may be expressed by a bacterium detailedherein. Furthermore, antigens for use in the invention are not limitedto those from pathogenic organisms. The selection and recombinantsynthesis of antigens has been previously described by Schodel (9) andCurtiss (10). Immunogenicity of the bacterium may be augmented and/ormodulated by constructing strains that also express sequences forcytokines, adjuvants, and other immunomodulators.

Some examples of microorganisms useful as a source for antigen arelisted below. These may include microoganisms for the control of plaguecaused by Yersinia pestis and other Yersinia species such as Y.pseudotuberculosis and Y. enterocolitica, for the control of gonorrheacaused by Neisseria gonorrhoea, for the control of syphilis caused byTreponema pallidum, and for the control of venereal diseases as well aseye infections caused by Chlamydia trachomatis. Species of Streptococcusfrom both group A and group B, such as those species that cause sorethroat or heart diseases, Erysipelothrix rhusiopathiae, Neisseriameningitidis, Mycoplasma pneumoniae and other Mycoplasma-species,Hemophilus influenza, Bordetella pertussis, Mycobacterium tuberculosis,Mycobacterium leprae, other Bordetella species, Escherichia coli,Streptococcus equi, Streptococcus pneumoniae, Brucella abortus,Pasteurella hemolytica and P. multocida, Vibrio cholera, Shigellaspecies, Borrellia species, Bartonella species, Heliobacter pylori,Campylobacter species, Pseudomonas species, Moraxella species, Brucellaspecies, Francisella species, Aeromonas species, Actinobacillus species,Clostridium species, Rickettsia species, Bacillus species, Coxiellaspecies, Ehrlichia species, Listeria species, and Legionella pneumophilaare additional examples of bacteria within the scope of this inventionfrom which antigen nucleic acid sequences could be obtained. Viralantigens may also be used. Viral antigens may be used in antigendelivery microorganisms directed against viruses, either DNA or RNAviruses, for example from the classes Papovavirus, Adenovirus,Herpesvirus, Poxvirus, Parvovirus, Reovirus, Picornavirus, Myxovirus,Paramyxovirus, Flavivirus or Retrovirus. Antigens may also be derivedfrom pathogenic fungi, protozoa and parasites.

Certain embodiments encompass an allergen as an antigen. Allergens aresubstances that cause allergic reactions in a host that is exposed tothem. Allergic reactions, also known as Type I hypersensitivity orimmediate hypersensitivity, are vertebrate immune responsescharacterized by IgE production in conjunction with certain cellularimmune reactions. Many different materials may be allergens, such asanimal dander and pollen, and the allergic reaction of individual hostswill vary for any particular allergen. It is possible to inducetolerance to an allergen in a host that normally shows an allergicresponse. The methods of inducing tolerance are well-known and generallycomprise administering the allergen to the host in increasing dosages.

It is not necessary that the vector comprise the complete nucleic acidsequence of the antigen. It is only necessary that the antigen sequenceused be capable of eliciting an immune response. The antigen may be onethat was not found in that exact form in the parent organism. Forexample, a sequence coding for an antigen comprising 100 amino acidresidues may be transferred in part into a recombinant bacterium so thata peptide comprising only 75, 65, 55, 45, 35, 25, 15, or even 10, aminoacid residues is produced by the recombinant bacterium. Alternatively,if the amino acid sequence of a particular antigen or fragment thereofis known, it may be possible to chemically synthesize the nucleic acidfragment or analog thereof by means of automated nucleic acid sequencesynthesizers, PCR, or the like and introduce said nucleic acid sequenceinto the appropriate copy number vector.

In another alternative, a vector may comprise a long sequence of nucleicacid encoding several nucleic acid sequence products, one or all ofwhich may be antigenic. In some embodiments, a vector of the inventionmay comprise a nucleic acid sequence encoding at least one antigen, atleast two antigens, at least three antigens, or more than threeantigens. These antigens may be encoded by two or more open readingframes operably linked to be expressed coordinately as an operon,wherein each antigen is synthesized independently. Alternatively, thetwo or more antigens may be encoded by a single open reading frame suchthat the antigens are synthesized as a fusion protein.

In certain embodiments, an antigen of the invention may comprise a Bcell epitope or a T cell epitope. Alternatively, an antigen to which animmune response is desired may be expressed as a fusion to a carrierprotein that contains a strong promiscuous T cell epitope and/or servesas an adjuvant and/or facilitates presentation of the antigen toenhance, in all cases, the immune response to the antigen or itscomponent part. This can be accomplished by methods known in the art.Fusion to tenus toxin fragment C, CT-B, LT-B and hepatitis virus B coreare particularly useful for these purposes, although other epitopepresentation systems are well known in the art.

In further embodiments, a nucleic acid sequence encoding an antigen ofthe invention may comprise a secretion signal. In other embodiments, anantigen of the invention may be toxic to the recombinant bacterium.

ii. Promoter Regulated by Repressor

The vector comprises a nucleic acid sequence encoding at least oneantigen operably-linked to a promoter regulated by the repressor,encoded by a chromosomally integrated nucleic acid sequence. One ofskill in the art would recognize, therefore, that the selection of arepressor dictates, in part, the selection of the promoteroperably-linked to a nucleic acid sequence encoding an antigen ofinterest. For instance, if the repressor is Lacl, then the promoter maybe selected from the group consisting of Lacl responsive promoters, suchas P_(trc), P_(lac), P_(T7lac) and P_(tac). If the repressor is C2, thenthe promoter may be selected from the group consisting of C2 responsivepromoters, such as P22 promoters P_(L) and P_(R). If the repressor isC1, then the promoter may be selected from the group consisting of C1responsive promoters, such as λ promoters P_(L) and P_(R).

In each embodiment herein, the promoter regulates expression of anucleic acid sequence encoding the antigen, such that expression of thenucleic acid sequence encoding an antigen is repressed when therepressor is synthesized (i.e. during in vitro growth of the bacterium),but expression of the nucleic acid sequence encoding an antigen is highwhen the repressor is not synthesized (i.e. in an animal or human host).Generally speaking, the concentration of the repressor will decreasewith every cell division after expression of the nucleic acid sequenceencoding the repressor ceases. In some embodiments, the concentration ofthe repressor decreases enough to allow high level expression of thenucleic acid sequence encoding an antigen after about 2, 3, 4, 5, 6, 7,8, 9, 10, 11, or 12 divisions of the bacterium. In an exemplaryembodiment, the concentration of the repressor decreases enough to allowhigh level expression of the nucleic acid sequence encoding an antigenafter about 5 divisions of the bacterium in an animal or human host.

In certain embodiments, the promoter may comprise other regulatoryelements. For instance, the promoter may comprise lacO if the repressoris Lacl. This is the case with the lipoprotein promoter P_(lpp) that isregulated by Lacl since it possesses the Lacl binding domain lacO.

In one embodiment, the repressor is a Lacl repressor and the promoter isP_(trc).

iii. Expression of the Nucleic Acid Sequence Encoding an Antigen

As detailed above, generally speaking the expression of the nucleic acidsequence encoding the antigen should be repressed when the repressor issynthesized. For instance, if the repressor is synthesized during invitro growth of the bacterium, expression of the nucleic acid sequenceencoding the antigen should be repressed. Expression may be “repressed”or “partially repressed” when it is about 50%, 45%, 40%, 35%, 30%, 25%,20%, 15%, 10%, 5%, 1%, or even less than 1% of the expression undernon-repressed conditions. Thus although the level of expression underconditions of “complete repression” might be exceeding low, it is likelyto be detectable using very sensitive methods since repression can neverby absolute.

Conversely, the expression of the nucleic acid sequence encoding theantigen should be high when the expression of the nucleic acid sequenceencoding the repressor is repressed. For instance, if the nucleic acidsequence encoding the repressor is not expressed during growth of therecombinant bacterium in the host, the expression of the nucleic acidsequence encoding the antigen should be high. As used herein, “highlevel” expression refers to expression that is strong enough to elicitan immune response to the antigen. Consequently, the copy numbercorrelating with high level expression can and will vary depending onthe antigen and the type of immune response desired. Methods ofdetermining whether an antigen elicits an immune response such as bymeasuring antibody levels or antigen-dependant T cell populations orantigen-dependant cytokine levels are known in the art, and methods ofmeasuring levels of expression of antigen encoding sequences bymeasuring levels of mRNA transcribed or by quantitating the level ofantigen synthesis are also known in the art. For more details, see theexamples.

(c) Crp Cassette

In some embodiments, a recombinant bacterium of the invention may alsocomprise a ΔP_(crp)::TT araC P_(BAD) crp deletion-insertion mutation.Since the araC P_(BAD) cassette is dependent both on the presence ofarabinose and the binding of the catabolite repressor protein Crp, aΔP_(crp)::TT araC P_(BAD) crp deletion-insertion mutation may beincluded as an additional means to reduce expression of any nucleic acidsequence under the control of the P_(BAD) promoter. This means that whenthe bacterium is grown in a non-permissive environment (i.e. noarabinose) both the repressor itself and the Crp protein cease to besynthesized, consequently eliminating both regulating signals for thearaC P_(BAD) regulated nucleic acid sequence. This double shut off ofaraC P_(BAD) may constitute an additional safety feature ensuring thegenetic stability of the desired phenotypes.

Generally speaking, the activity of the Crp protein requires interactionwith cAMP, but the addition of glucose, which may inhibit synthesis ofcAMP, decreases the ability of the Crp protein to regulate transcriptionfrom the araC P_(BAD) promoter. Consequently, to avoid the effect ofglucose on cAMP, glucose may be substantially excluded from the growthmedia, or variants of crp may be isolated that synthesize a Crp proteinthat is not dependent on cAMP to regulate transcription from P_(BAD).This strategy may also be used in other systems responsive to Crp, suchas the systems responsive to rhamnose and xylose described above.

(d) Attenuation

In each of the above embodiments, a recombinant bacterium of theinvention capable of regulated expression may also be attenuated.“Attenuated” refers to the state of the bacterium wherein the bacteriumhas been weakened from its wild type fitness by some form of recombinantor physical manipulation. This includes altering the genotype of thebacterium to reduce its ability to cause disease. However, thebacterium's ability to colonize the gut (in the case of Salmonella) andinduce immune responses is, preferably, not substantially compromised.

In an exemplary embodiment, a recombinant bacterium may be attenuated asdescribed in section II below. In which case, both regulated attenuationand regulated expression of an antigen encoding sequence may bedependent upon an arabinose regulatable system. Consequently, theconcentration of arabinose needed for optimal expression of theregulated antigen encoding sequence may not be the same as theconcentration for optimal expression of attenuation. In an exemplaryembodiment, the concentration of arabinose for the optimization of bothregulated attenuation and regulated expression of sequences encodingantigen will be substantially the same.

Accordingly, the promoter and/or the nucleic acid sequence encoding anattenuation protein may be modified to optimize the system. Methods ofmodification are detailed above. Briefly, for example, the SD ribosomebinding sequence may be altered, and/or the start codon may be alteredfrom ATG to GTG for the nucleic acid sequences fur and phoPQ, so thatthe production levels of Fur and PhoPQ are optimal for both theregulated attenuation phenotype and the regulated expression whengrowing strains with a given concentration of arabinose. One of skill inthe art will appreciate that other nucleic acid sequences, in additionto fur and phoPQ, may also be altered as described herein in combinationwith other well-known protocols. In addition, these attenuating nucleicacid sequences may be regulated by other systems using well-establishedprotocols known to one of skill in the art. For example, they may beregulated using with promoters dependent on addition of maltose,rhamnose, or xylose rather than arabinose.

Other methods of attenuation are known in the art. For instance,attenuation may be accomplished by altering (e.g., deleting) nativenucleic acid sequences found in the wild type bacterium. For instance,if the bacterium is Salmonella, non-limiting examples of nucleic acidsequences which may be used for attenuation include: a pab nucleic acidsequence, a pur nucleic acid sequence, an aro nucleic acid sequence,asd, a dap nucleic acid sequence, nadA, pncB, galE, pmi, fur, rpsL,ompR, htrA, hemA, cdt, cya, crp, dam, phoP, phoQ, rfc, poxA, galU, mviA,sodC, recA, ssrA, sirA, inv, hilA, rpoE, flgM, tonB, slyA, and anycombination thereof. Exemplary attenuating mutations may be aroA, aroC,aroD, cdt, cya, crp, phoP, phoQ, ompR, galE, and htrA.

In certain embodiments, the above nucleic acid sequences may be placedunder the control of a sugar regulated promoter wherein the sugar ispresent during in vitro growth of the recombinant bacterium, butsubstantially absent within an animal or human host. The cessation intranscription of the nucleic acid sequences listed above would thenresult in attenuation and the inability of the recombinant bacterium toinduce disease symptoms.

In another embodiment, the recombinant bacterium may contain one and insome embodiments, more than one, deletion and/or deletion-insertionmutation present in the strains listed in Table 1 above. Furthermore,suicide vectors, as listed in Table 2, and ss described in the Examplesbelow, along with other plasmid vectors, may be used to introduce thesedeletion and deletion-insertion mutations into strains during theirconstruction.

TABLE 2 Plasmid Properties Suicide vector pMEG-375 sacRB mobRP4 R6K oriCm^(r) Ap^(r) pMEG-443 ΔasdA16 pRE112 SacB mobRP4 R6K ori Cm^(r) pYA3438ΔpabB232 in pMEG-375 pYA3485 ΔaraE25 in pMEG-375 pYA3599 ΔaraBAD23 inpMEG-375 pYA3629 Δ(gmd-fcl)-26 in pMEG-375 pYA3652 ΔendA2311 in pMEG-375pYA3679 ΔrelA1123 in pMEG-375 pYA3722 ΔP_(fur33)::TT araC P_(BAD) fur inpMEG-375 pYA3723 ΔP_(phoPQ107)::TT araC P_(BAD) phoPQ in pRE112 pYA3735ΔP_(rpoS183)::TT araC P_(BAD) rpoS in pRE112 pYA3736 ΔasdA33 in pRE112pYA3737 ΔasdA18::TT araC P_(BAD) P22 c2 in pRE112 pYA3783 pRE112 derivedsuicide vector to generate GTG-lacI, ΔendA19::araC P_(BAD) lacI TTmutation, Cm^(r) pYA3784 pRE112 derived suicide vector to generateGTG-lacI, ΔrelA196::araC P_(BAD) lacI TT mutation, Cm^(r) pYA3832ΔP_(crp527)::TT araC P_(BAD) crp in pRE112 pYA3871 pRE112 derivedsuicide vector to generate ATG-lacI, ΔendA20::araC P_(BAD) lacI TTmutation, Cm^(r) pYA3879 pRE112 derived suicide vector to generateATG-lacI, ΔrelA197::araC P_(BAD) lacI TT mutation, Cm^(r) pYA4062ΔP_(phoPQ173)::TT araC P_(BAD) phoPQ in pRE112 pYA4063 pRE112 derivedsuicide vector to generate improved SD ATG-lacI, ΔendA21::araC P_(BAD)lacI (improved codon) TT mutation, Cm^(r) pYA4064 pRE112 derived suicidevector to generate codon optimized lacI, ΔrelA198::araC P_(BAD) lacI TTmutation, Cm^(r) pYA4109 ΔP_(phoPQ177)::TT araC P_(BAD) phoPQ in pRE112pYA4138 ΔasdA27::TT araC P_(BAD) P22 c2 in pRE112 pYA4177 ΔasdA21::TTaraC P_(BAD) P22 c2 in pRE112 pYA4180 ΔP_(fur77)::TT araC P_(BAD) fur inpRE112 pYA4181 ΔP_(fur81)::TT araC P_(BAD) fur in pRE112 pYA4213ΔasdA20::TT araC P_(BAD) P22 c2 in pRE112 pYA4235 ΔP_(murA12)::TT araCP_(BAD) murA in pRE112 pYA4280 Δ(araC P_(BAD))-5: P22 P_(R) araBAD inpRE112 pYA4343 ΔP_(phoPQ176)::TT araC P_(BAD) phoPQ in pRE112 pYA4344ΔP_(phoPQ175)::TT araC P_(BAD) phoPQ in pRE112 pYA4345 ΔP_(phoPQ174)::TTaraC P_(BAD) phoPQ in pRE112 Recombinant vector pGEM-3Z Ap^(r), Cloningvector, pUC ori pYA3342 pBR ori Asd⁺, 3012 bp pYA3450 p15A ori araC ^(§)P_(BAD) SD-ATG asdA pYA3493 pBR ori bla SS, Asd⁺, 3113 bp, pYA3530 p15Aori araC ^(§) P_(BAD) SD-GTG asdA pYA3552 Asd⁺ vector expression gfp3,pBR ori pYA3620 pBR ori Asd⁺, bla SS bla CT, 3169 bp pYA3624 Plasmidwith tightly regulated araC P_(BAD) cassette, p15A ori pYA3634 pBR oribla SS, Asd⁺ PspA Rx1 aa 3-257 pYA3635 pBR ori bla SS, Asd⁺ PspA Rx1 aa3-257 (codon optimized) pYA3681 pBR ori araC P_(BAD) SD-GTG asdA SD-GTGmurA P22 P_(R) anti-sense mRNA pYA3685 pBR ori araC P_(BAD) SD-GTG asdASD-GTG murA P22 P_(R) anti-sense mRNA, rPspA Rx1 pYA3698 pGEM-3Z with T4ipIII transcription terminator, Ap^(r) pYA3699 pGEM-3Z with araC P_(BAD)cassette, Ap^(r) pYA3700 AraC P_(BAD) TT cassette plasmid, Ap^(r)pYA3782 P_(BAD) regulated lacI (GTG) in pYA3700-PCR-Topo-Blunt IIpYA3789 Runaway vector harboring araC P_(BAD) P22 c2, GTG-MurA⁺,GTG-Asd⁺, P_(trc), pSC101 ori, pUC ori pYA3856 pBAD-HisA with GTG startcodon lacI, His-tagged, Ap^(r) pYA4050 pUC ori araC P_(BAD) SD-GTG murASD-GTF asd, P22P_(R) antisense RNA with DNA nuclear Targeting sequenceand poly A from SV40, 6941 bp pYA4088 pBR ori bla SS, Asd⁺ PspA Rx1 aa3-285 (codon optimized) pYA4090 Asd⁺ vector, P_(trc) gfp3, pBR ori p =plasmid P = promoter SD = Shine-Dalgarno sequence araC ^(§) P_(BAD) = E.coli B/r aa = amino acid SS = signal sequence Ap^(r) = ampicillinresistance Cm^(r) = chloramphenicol resistance

The bacterium may also be modified to create a balanced-lethalhost-vector system, although other types of systems may also be used(e.g., creating complementation heterozygotes). For the balanced-lethalhost-vector system, the bacterium may be modified by manipulating itsability to synthesize various essential constituents needed forsynthesis of the rigid peptidoglycan layer of its cell wall. In oneexample, the constituent is diaminopimelic acid (DAP). Various enzymesare involved in the eventual synthesis of DAP. In one example, thebacterium is modified by using a ΔasdA mutation to eliminate thebacterium's ability to produce β-aspartate semialdehyde dehydrogenase,an enzyme essential for the synthesis of DAP. One of skill in the artcan also use the teachings of U.S. Pat. No. 6,872,547 for other types ofmutations of nucleic acid sequences that result in the abolition of thesynthesis of DAP. These nucleic acid sequences may include, but are notlimited to, dapA, dapB, dapC, dapD, dapE, dapF, and asd. Othermodifications that may be employed include modifications to abacterium's ability to synthesize D-alanine or to synthesize D-glutamicacid (e.g., ΔmurI mutations), which are both unique constituents of thepeptidoglycan layer of the bacterial cell wall

Yet another balanced-lethal host-vector system comprises modifying thebacterium such that the synthesis of an essential constituent of therigid layer of the bacterial cell wall is dependent on a nutrient (e.g.,arabinose) that can be supplied during the growth of the microorganism.For example, a bacterium may be comprise the ΔP_(murA)::TT araC P_(BAD)murA deletion-insertion mutation. This type of mutation makes synthesisof muramic acid (another unique essential constituent of thepeptidoglycan layer of the bacterial cell wall) dependent on thepresence of arabinose that can be supplied during growth of thebacterium in vitro.

However, when arabinose is absent as it is in an animal or human host,the essential constitutent of the peptidoglycan layer of the cell wallis not synthesized. This mutation represents an arabinose dependantlethal mutation. In the absence of arabinose, synthesis of muramic acidceases and lysis of the bacterium occurs because the peptidoglycan layerof the cell wall is not synthesized. It is not possible to generateΔmurA mutations because they are lethal. The necessary nutrient, aphosphorylated muramic acid, can not be exogenously supplied becauseenteric bacteria cannot take the nutrient up from the media. Recombinantbacteria with a ΔP_(murA)::TT araC P_(BAD) murA deletion-insertionmutation grown in the presence of arabinose exhibit effectivecolonization of effector lymphoid tissues after oral vaccination priorto undergoing lysis due to the inability to synthesize muramic acid.

Similarly, various embodiments may comprise the araC P_(BAD) c2 cassetteinserted into the asd nucleic acid sequence that encodes aspartatesemialdehyde dehydrogenase. Since the araC nucleic acid sequence istranscribed in a direction that could lead to interference in theexpression of adjacent nucleic acid sequences and adversely affectvaccine strain performance, a transcription termination (TT) sequence isgenerally inserted 3′ to the araC nucleic acid sequence. The chromosomalasd nucleic acid sequence is typically inactivated to enable use ofplasmid vectors encoding the wild-type asd nucleic acid sequence in thebalanced lethal host-vector system. This allows stable maintenance ofplasmids in vivo in the absence of any drug resistance attributes thatare not permissible in live bacterial vaccines. In some of theseembodiments, the wild-type asd nucleic acid sequence may be encoded bythe vector described above. The vector enables the regulated expressionof an antigen encoding sequence through the repressible promoter. Forinstance, in one embodiment shown in FIG. 13, pYA3634 has the Asd vectorspecifying the antigen PspA Rx1.

In one embodiment shown in FIG. 4, ΔasdA271::TT araC P_(BAD) c2 has animproved SD sequence and a codon optimized c2 nucleic acid sequence(FIG. 5). The C2 repressor synthesized in the presence of arabinose isused to repress nucleic acid sequence expression from P22 P_(R) andP_(L) promoters. In another embodiment shown in FIG. 4, ΔasdA27::TT araCP_(BAD) c2 (the preferred embodiment) has the 1104 base-pair asd nucleicacid sequence deleted (1 to 1104, but not including the TAG stop codon)and the 1989 base-pair fragment containing T4 iplll TT araC P_(BAD) c2inserted. The c2 nucleic acid sequence in ΔasdA27::TT araC P_(BAD) c2has a SD sequence that was optimized to TAAGGAGGT. It also has animproved P_(BAD) promoter such that the −10 sequence is improved fromTACTGT to TATAAT. Furthermore, it has a codon optimized c2 nucleic acidsequence, in which the second codon was modified from AAT to AAA (FIG.6).

In further embodiments, the bacterium may be attenuated by regulatingthe murA nucleic acid sequence encoding the first enzyme in muramic acidsynthesis and the asd nucleic acid sequence essential for DAP synthesis.These embodiments may comprise the chromosomal deletion-insertionmutations ΔasdA19::TT araC P_(BAD) c2 or ΔasdA27::TT araC P_(BAD) c2 andΔP_(murA7)::TT araC P_(BAD) murA or ΔP_(murA12)::TT araC P_(BAD) murA.This host-vector grows in LB broth with 0.1% L-arabinose, but is unableto grow in or on media devoid of arabinose since it undergoes cellwall-less death by lysis. In some embodiments of the invention, therecombinant bacterium may comprise araBAD and araE mutations to precludebreakdown and leakage of internalized arabinose such that asd and murAnucleic acid sequence expression continues for a cell division or twoafter oral immunization into an environment that is devoid of externalarabinose. (For example a strain with the ΔP_(murA7)::TT araC P_(BAD)murA deletion-insertion mutation undergoes about two cell divisions andthen commences to lyse in media made of mouse or chicken feed or chickenbreast meat, unless they are supplemented with arabinose.) Either GTG orTTG start codons for the murA and asd nucleic acid sequences areimportant to decrease translation efficiency on multi-copy plasmids.This embodiment is illustrated by FIG. 14, which shows the plasmidvector pYA3681. This vector contains the murA nucleic acid sequence(with altered start codon sequences to decrease translation efficiency)under the control of an araC P_(BAD) promoter. Also the second nucleicacid sequence under the direction of this promoter is the asd nucleicacid sequence (with altered start codon sequences to decreasetranslation efficiency). The P22 P_(R) promoter is in the anti-sensedirection of both the asd nucleic acid sequence and the murA nucleicacid sequence. The P22 P_(R) is repressed by the C2 repressor madeduring growth of the strain in media with arabinose (due to theΔasdA19::TT araC P_(BAD) c2 deletion-insertion). However C2concentration decreases due to cell division in vivo to cause P_(R)directed synthesis of anti-sense mRNA to further block translation ofasd and murA mRNA. The araC P_(BAD) sequence is also not from E. coliB/r as originally described (5) but represents a sequence derived fromE. coli K-12 strain χ289 with tighter control and less leakiness in theabsence of arabinose. In the preferred embodiment, transcriptionterminators (TT) flank all of the domains for controlled lysis,replication, and expression so that expression in one domain does notaffect the activities of another domain. As a safety feature, theplasmid asd nucleic acid sequence does not replace the chromosomal asdmutation since they have a deleted sequence in common, consequently, theE. coli murA nucleic acid sequence was used in the plasmid instead ofusing the Salmonella murA nucleic acid sequence. The recombinantbacterium of this embodiment is avirulent at oral doses in excess of 10⁹CFU to BALB/c mice. In addition to being fully attenuated, thisconstruction exhibits complete biological containment with no in vivorecombinant bacteria survivors detectable after 21 days and norecombinant bacteria survivors during or after excretion. This propertyenhances vaccine safety and minimizes the potential for vaccination ofindividuals not intended for vaccination.

II. Regulated Attenuation

The present invention also encompasses a recombinant bacterium capableof regulated attenuation. Generally speaking, the bacterium comprises achromosomally integrated regulatable promoter. The promoter replaces thenative promoter of, and is operably linked to, at least one nucleic acidsequence encoding an attenuation protein, such that the absence of thefunction of the protein renders the bacterium attenuated. In someembodiments, the promoter is modified to optimize the regulatedattenuation.

In each of the above embodiments described herein, more than one methodof attenuation may be used. For instance, a recombinant bacterium of theinvention may comprise a regulatable promoter chromosomally integratedso as to replace the native promoter of, and be operably linked to, atleast one nucleic acid sequence encoding an attenuation protein, suchthat the absence of the function of the protein renders the bacteriumattenuated, and the bacterium may comprise another method of attenuationdetailed in section I above.

(a) Attenuation Protein

Herein, “attenuation protein” is meant to be used in its broadest senseto encompass any protein the absence of which attenuates a bacterium.For instance, in some embodiments, an attenuation protein may be aprotein that helps protect a bacterium from stresses encountered in thegastrointestinal tract or respiratory tract. Non-limiting examples maybe the RpoS, PhoPQ, OmpR, Fur, and Crp proteins. In other embodiments,the protein may be a necessary component of the cell wall of thebacterium, such as the protein encoded by murA. In still otherembodiments, the protein may be listed in Section I(d) above.

The native promoter of at least one, two, three, four, five, or morethan five attenuation proteins may be replaced by a regulatable promoteras described herein. In one embodiment, the promoter of one of theproteins selected from the group comprising RpoS, PhoPQ, OmpR, Fur, andCrp may be replaced. In another embodiment, the promoter of two, three,four or five of the proteins selected from the group comprising RpoS,PhoPQ, OmpR, Fur, and Crp may be replaced.

If the promoter of more than one attenuation protein is replaced, eachpromoter may be replaced with a regulatable promoter, such that theexpression of each attenuation protein encoding sequence is regulated bythe same compound or condition. Alternatively, each promoter may bereplaced with a different regulatable promoter, such that the expressionof each attenuation protein encoding sequence is regulated by adifferent compound or condition such as by the sugars arabinose,maltose, rhamnose or xylose.

(b) Regulatable Promoter

The native promoter of a nucleic acid encoding an attenuation protein isreplaced with a regulatable promoter operably linked to the nucleic acidsequence encoding an attenuation protein. The term “operably linked,” isdefined above.

The regulatable promoter used herein generally allows transcription ofthe nucleic acid sequence encoding the attenuation protein while in apermissive environment (i.e. in vitro growth), but cease transcriptionof the nucleic acid sequence encoding an attenuation protein while in anon-permissive environment (i.e. during growth of the bacterium in ananimal or human host). For instance, the promoter may be responsive to aphysical or chemical difference between the permissive andnon-permissive environment. Suitable examples of such regulatablepromoters are known in the art and detailed above.

In some embodiments, the promoter may be responsive to the level ofarabinose in the environment, as described above. In other embodiments,the promoter may be responsive to the level of maltose, rhamnose, orxylose in the environment, as described above. The promoters detailedherein are known in the art, and methods of operably linking them to anucleic acid sequence encoding an attenuation protein are known in theart.

In certain embodiments, a recombinant bacterium of the invention maycomprise any of the following: ΔP_(fur)::TT araC P_(BAD) fur,ΔP_(crp)::TT araC P_(BAD) crp, ΔP_(phoPQ)::TT araC P_(BAD) phoPQ, or acombination thereof. (P stands for promoter and TT stands fortranscription terminator). Growth of such strains in the presence ofarabinose leads to transcription of the fur, phoPQ, and/or crp nucleicacid sequences, but nucleic acid sequence expression ceases in a hostbecause there is no free arabinose. Attenuation develops as the productsof the fur, phoPQ, and/or the crp nucleic acid sequences are diluted ateach cell division. Strains with the ΔP_(fur) and/or the ΔP_(phoPQ)mutations are attenuated at oral doses of 10⁹ CFU, even in three-weekold mice at weaning. Generally speaking, the concentration of arabinosenecessary to induce expression is typically less than about 2%. In someembodiments, the concentration is less than about 1.5%, 1%, 0.5%, 0.2%,0.1%, or 0.05%. In certain embodiments, the concentration may be about0.04%, 0.03%, 0.02%, or 0.01%. In an exemplary embodiment, theconcentration is about 0.05%. Higher concentrations of arabinose orother sugars may lead to acid production during growth that may inhibitdesirable cell densities. The inclusion of mutations such as ΔaraBAD ormutations that block the uptake and/or breakdown of maltose, rhamnose,or xylose, however, may prevent such acid production and enable use ofhigher sugar concentrations with no ill effects.

When the regulatable promoter is responsive to arabinose, the onset ofattenuation may be delayed by including additional mutations, such asΔaraBAD23, which prevents use of arabinose retained in the cellcytoplasm at the time of oral immunization, and/or ΔaraE25 that enhancesretention of arabinose. Thus, inclusion of these mutations may bebeneficial in at least two ways: first, enabling higher culturedensities, and second enabling a further delay in the display of theattenuated phenotype that may result in higher densities in effectorlymphoid tissues to further enhance immunogenicity.

(c) Modifications

Attenuation of the recombinant bacterium may be optimized by modifyingthe nucleic acid sequence encoding an attenuation protein and/orpromoter. Methods of modifying a promoter and/or a nucleic acid sequenceencoding an attenuation protein are the same as those detailed abovewith respect to repressors in Section I.

In some embodiments, more than one modification may be performed tooptimize the attenuation of the bacterium. For instance, at least one,two, three, four, five, six, seven, eight or nine modifications may beperformed to optimize the attenuation of the bacterium.

In various exemplary embodiments of the invention, the SD sequencesand/or the start codons for the fur and/or the phoPQ virulence nucleicacid sequences may be altered so that the production levels of thesenucleic acid products are optimal for regulated attenuation. FIG. 8depicts ΔP_(fur77)::TT araC P_(BAD) fur, whose start codon is changedfrom ATG to GTG, and ΔP_(fur81)::TT araC P_(BAD) fur, that has aweakened SD sequence as well as the start codon changed from ATG to GTG.FIG. 9 depicts ΔP_(phopQ173)::TT araC P_(BAD) phoPQ, that hasmodifications to the start codon as well as the second codon, which waschanged from ATG to GTG. FIG. 9 also depicts ΔP_(phoPQ177)::TT araCP_(BAD) phOPQ, wherein the SD sequence has been changed to the weakerAAGG sequence, the start codon was modified, and the second codon wasmodified from ATG to GTG.

(d) Crp Cassette

In some embodiments, a recombinant bacterium of the invention may alsocomprise a ΔP_(crp)::TT araC P_(BAD) crp deletion-insertion mutation(FIG. 7), as described above. Since the araC P_(BAD) cassette isdependent both on the presence of arabinose and the binding of thecatabolite repressor protein Crp, a ΔP_(crp)::TT araC P_(BAD) crpdeletion-insertion mutation may be included as an additional control onthe expression of the nucleic acid sequence encoding an attenuationprotein.

Generally speaking, the activity of the Crp protein requires interactionwith cAMP, but the addition of glucose, which may inhibit synthesis ofcAMP, decreases the ability of the Crp protein to regulate transcriptionfrom the araC P_(BAD) promoter. Consequently, to avoid the effect ofglucose on cAMP, glucose may be substantially excluded from the growthmedia, or variants of crp may be isolated that synthesize a Crp proteinthat is not dependent on cAMP to regulate transcription from P_(BAD).This strategy may also be used in other systems responsive to Crp, suchas the systems responsive to rhamnose and xylose described above

(e) Regulated Expression

In each of the above embodiments, a bacterium capable of regulatedattenuation may also be capable of regulated expression of at least onenucleic acid encoding an antigen as detailed in section I above.

For instance, various embodiments of the present invention may encompassa recombinant pathogenic Enterobacteriaceae species comprisingdeletion-insertion mutations conferring regulated attenuation andregulated expression of a nucleic acid sequence encoding an antigen. Insome embodiments, the recombinant bacterium may further comprise atleast one chromosomal nucleic acid sequence containing a mutationconferring a lethal phenotype. The mutated chromosomal nucleic acidsequence may be complemented by a plasmid vector containing a functionalnucleic acid sequence corresponding to the mutated chromosomal nucleicacid sequence.

III. Vaccine Compositions and Administration

A recombinant bacterium of the invention may be administered to a hostas a vaccine composition. As used herein, a vaccine composition is acomposition designed to elicit an immune response to the recombinantbacterium, including any antigens that may be expressed by thebacterium. In an exemplary embodiment, the immune response isprotective, as described above. Immune responses to antigens are wellstudied and widely reported. A survey of immunology is given by aul, WE, Stites D et. al. and Ogra P L. et. al. (11-13). Mucosal immunity isalso described by Ogra P L et. al. (14).

Vaccine compositions of the present invention may be administered to anyhost capable of mounting an immune response. Such hosts may include allvertebrates, for example, mammals, including domestic animals,agricultural animals, laboratory animals, and humans, and variousspecies of birds, including domestic birds and birds of agriculturalimportance. Preferably, the host is a warm-blooded animal. The vaccinecan be administered as a prophylactic or for treatment purposes.

In exemplary embodiments, the recombinant bacterium is alive whenadministered to a host in a vaccine composition of the invention.Suitable vaccine composition formulations and methods of administrationare detailed below.

(a) Vaccine Composition

A vaccine composition comprising a recombinant bacterium of theinvention may optionally comprise one or more possible additives, suchas carriers, preservatives, stabilizers, adjuvants, and othersubstances.

In one embodiment, the vaccine comprises an adjuvant. Adjuvants, such asaluminum hydroxide or aluminum phosphate, are optionally added toincrease the ability of the vaccine to trigger, enhance, or prolong animmune response. In exemplary embodiments, the use of a live attenuatedrecombinant bacterium may act as a natural adjuvant. The vaccinecompositions may further comprise additional components known in the artto improve the immune response to a vaccine, such as T cellco-stimulatory molecules or antibodies, such as anti-CTLA4. Additionalmaterials, such as cytokines, chemokines, and bacterial nucleic acidsequences naturally found in bacteria, like CpG, are also potentialvaccine adjuvants.

In another embodiment, the vaccine may comprise a pharmaceutical carrier(or excipient). Such a carrier may be any solvent or solid material forencapsulation that is non-toxic to the inoculated host and compatiblewith the recombinant bacterium. A carrier may give form or consistency,or act as a diluent. Suitable pharmaceutical carriers may include liquidcarriers, such as normal saline and other non-toxic salts at or nearphysiological concentrations, and solid carriers not used for humans,such as talc or sucrose, or animal feed. Carriers may also includestabilizing agents, wetting and emulsifying agents, salts for varyingosmolarity, encapsulating agents, buffers, and skin penetrationenhancers. Carriers and excipients as well as formulations forparenteral and nonparenteral drug delivery are set forth in Remington'sPharmaceutical Sciences 19th Ed. Mack Publishing (1995). When used foradministering via the bronchial tubes, the vaccine is preferablypresented in the form of an aerosol.

Care should be taken when using additives so that the live recombinantbacterium is not killed, or have its ability to effectively colonizelymphoid tissues such as the GALT, NALT and BALT compromised by the useof additives. Stabilizers, such as lactose or monosodium glutamate(MSG), may be added to stabilize the vaccine formulation against avariety of conditions, such as temperature variations or a freeze-dryingprocess.

The dosages of a vaccine composition of the invention can and will varydepending on the recombinant bacterium, the regulated antigen, and theintended host, as will be appreciated by one of skill in the art.Generally speaking, the dosage need only be sufficient to elicit aprotective immune response in a majority of hosts. Routineexperimentation may readily establish the required dosage. Typicalinitial dosages of vaccine for oral administration could be about 1×10⁷to 1×10¹⁰ CFU depending upon the age of the host to be immunized.Administering multiple dosages may also be used as needed to provide thedesired level of protective immunity.

(b) Methods of Administration

In order to stimulate a preferred response of the GALT, NALT or BALTcells, administration of the vaccine composition directly into the gut,nasopharynx, or bronchus is preferred, such as by oral administration,intranasal administration, gastric intubation or in the form ofaerosols, although other methods of administering the recombinantbacterium, such as intravenous, intramuscular, subcutaneous injection orintramammary, intrapenial, intrarectal, vaginal administration, or otherparenteral routes, are possible.

In some embodiments, these compositions are formulated foradministration by injection (e.g., intraperitoneally, intravenously,subcutaneously, intramuscularly, etc.). Accordingly, these compositionsare preferably combined with pharmaceutically acceptable vehicles suchas saline, Ringer's solution, dextrose solution, and the like.

IV. Kits

The invention also encompasses kits comprising any one of thecompositions above in a suitable aliquot for vaccinating a host in needthereof. In one embodiment, the kit further comprises instructions foruse. In other embodiments, the composition is lyophilized such thataddition of a hydrating agent (e.g., buffered saline) reconstitutes thecomposition to generate a vaccine composition ready to administer,preferably orally.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention. Those of skill in the art should, however, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments that are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention, therefore all matter set forth or shown in the accompanyingdrawings is to be interpreted as illustrative and not in a limitingsense.

V. Methods of Use

A further aspect of the invention encompasses methods of using arecombinant bacterium of the invention. For instance, in one embodimentthe invention provides a method for modulating a host's immune system.The method comprises administering to the host an effective amount of acomposition comprising a recombinant bacterium of the invention. One ofskill in the art will appreciate that an effective amount of acomposition is an amount that will generate the desired immune response(e.g., mucosal, humoral or cellular). Methods of monitoring a host'simmune response are well-known to physicians and other skilledpractitioners. For instance, assays such as ELISA, and ELISPOT may beused. Effectiveness may be determined by monitoring the amount of theantigen of interest remaining in the host, or by measuring a decrease indisease incidence caused by a given pathogen in a host. For certainpathogens, cultures or swabs taken as biological samples from a host maybe used to monitor the existence or amount of pathogen in theindividual.

In another embodiment, the invention provides a method for eliciting animmune response against an antigen in a host. The method comprisesadministering to the host an effective amount of a compositioncomprising a recombinant bacterium of the invention

In still another embodiment, a recombinant bacterium of the inventionmay be used in a method for eliciting an immune response against apathogen in an individual in need thereof. The method comprisesadministrating to the host an effective amount of a compositioncomprising a recombinant bacterium as described herein. In a furtherembodiment, a recombinant bacterium described herein may be used in amethod for ameliorating one or more symptoms of an infectious disease ina host in need thereof. The method comprises administering an effectiveamount of a composition comprising a recombinant bacterium as describedherein.

REFERENCES (TO PREVIOUS ABOVE TEXT)

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EXAMPLES

The following examples illustrate various iterations of the invention.

Example 1: Regulated Expression of Antigen Encoding Nucleic AcidSequences

Antigens, delivered by recombinant attenuated Salmonella vaccine strains(RASVs), induce strong systemic and mucosal immune responses that aredependent on several factors including route of immunization [1, 2],expression level [3], cellular location [4], presentation [5], strainbackground [6, 7] and the inherent immunogenic properties of antigen.Generally, achieving maximal immune responses to the foreign antigen isdirectly correlated with the amount of the antigen produced [3, 8], thusit is important that the immunizing bacterial strain produce adequatelevels of antigen. However, for RASV, this need must be weighed againstthe fact that high level antigen production can be a drain on the energyresources of the bacterium, leading to reduced growth rates and acompromised ability to colonize and stimulate effector lymphoid tissues[9]. In addition, some antigens are inherently toxic to vaccine strainsfor other reasons, leading to a severe inhibition of growth rate andhost colonizing potential and, in some cases, death of the RASV.Sometimes, overexpression of foreign proteins can also result inmutations in the promoter or coding sequence of the nucleic acidsequence encoding the antigen, leading to unwanted changes in the levelof antigen synthesis or character, thus reducing or compromising thedesired immune response. Several approaches have been used to addressthis problem, including adopting in vivo inducible promoters, includingthose from the pagC [13], nirB [14], spvy and dps [15] nucleic acidsequences. In principle, the advantage of using inducible promoters isthat only low levels of antigen are produced during in vitro growth andthe initial stages of infection. These promoters then upregulate antigenexpression once the bacteria reach immunocompetent sites within thehost, thus inducing the desired antigen-specific immune response.However, inducible promoters, as they are presently known in the art,are often either too weak in vivo or too strong in vitro, and may belimited by the mode of attenuation [14, 16]. Therefore, there is a needin the art for a system with a promoter that is weakly active in vitro,is capable of strong expression in vivo and whose function is notinfluenced by the mode of attenuation.

In this example, the construction of such a system is describedutilizing the strong P_(trc) promoter for antigen expression andattenuated Salmonella enterica serovar Typhimurium strains expressingdifferent levels of Lacl under the control of an arabinose-regulatedpromoter. Two test antigens were used to evaluate the system. The greenfluorescent protein (GFP) was used for in vitro evaluation of the systemand the α-helical fragment of the Streptococcus pneumoniae pspA nucleicacid sequence [4, 17, 18] was used as the test antigen forimmunogenicity studies. Salmonella strains were constructed andevaluated for level of Lacl synthesis, antigen synthesis and the abilityto induce a protective immune response in mice.

Materials and Methods for Example 1

Bacterial Strains, Plasmids, Media and Growth Conditions:

Bacterial strains and plasmids used are listed in above Table 1 andTable 2, respectively. Bacteria were grown statically overnight at 37°C. in LB broth [19], 3XD broth, a buffered Casamino acids medium thatincludes glycerol as the carbon source [20] or nutrient broth (Difco) asindicated. The second day, the cultures were diluted 1:100 intopre-warmed media with aeration at 37° C. When required, antibiotics andsupplements were added at the following concentrations: chloramphenicol,30 μg/ml; Diaminopimelic acid (DAP), 50 μg/ml [21]; p-aminobenzoic acid(pABA), 10 μg/ml. LB agar without NaCl and containing 5% sucrose wasused for sacB nucleic acid sequence-based counter selection in allelicexchange experiments [22]. S. pneumoniae WU2 was cultured on brain heartinfusion agar containing 5% sheep blood or in Todd-Hewitt broth plus0.5% yeast extract[17].

General DNA Procedures:

DNA manipulations were carried out as described by Sambrook et al. [51].Transformation of bacterial strains was routinely done byelectroporation [52] using Nucleic acid sequence Pulser Xcell System(BioRad, Hercules, Calif.). Transformants containing Asd⁺ plasmids wereselected on LB agar plates without DAP. Only clones containing therecombinant plasmids were able to grow under these conditions. Suicidevector and P22-mediated transduction was used to generate defineddeletion/deletion-insertion mutation [53, 54]. Transfer of recombinantsuicide plasmids to Salmonella was accomplished by conjugation using E.coli χ7213 (Asd⁻) as the plasmid donor [48]. Bacteriophage P22HTint-mediated general transduction was performed by standard methods[55]. PCR amplification was employed to obtain DNA fragments for cloningand for verification of chromosomal deletion mutations. Nucleotidesequencing reactions were performed by the DNA lab in Arizona StateUniversity.

Construction of Plasmid pYA3700:

Plasmid pYA3700 carried a tightly regulated araC P_(BAD) TT cassette. Toconstruct this plasmid, two oligonucleotides,

(SEQ ID NO: 34) 5'-CCTGGTACCTAGGCCTCTAGATAAATAAAAGCAGTTTACAACTCCTAGAATTGTGAATATATTATCACAATTCTAGGATAGAATAATAA AAGATCTCTGCAGGGC-3'and its complement, corresponding to the T4 iplll transcriptionterminator [56] and additional enzyme site (underlined) were annealed,cut with KpnI-PstI, and cloned into pGEM3Z cut with the same enzymes tocreate plasmid pYA3698 (Table 2). The araC P_(BAD) cassette wasamplified using plasmid pYA3624 [57] as template with primer pair 1pBADaraCKpnI (5′-ΔGAGGTACCCTCGAGGCTAGCCCAAAAAAACGGG-3′) (SEQ ID NO:35)and 1pBADaraCXbaI (5′-TGGTCTAGAGTCAAGCCGTCAATTGTCTGATTCG-3′) (SEQ IDNO:36). The PCR fragment was cut with KpnI-XbaI and cloned into plasmidpGEM3Z to generate plasmid pYA3699 and into pYA3698 to generate theplasmid pYA3700.

Construction of Suicide Vector pYA3784:

The GTG-lacl nucleic acid sequences were amplified from the χ289 genomeusing the primer pairs, laclEcoRI-3′(5′-GGAATTCTCACTGCCCGCTTTCCAGTCGGG-3′) (SEQ ID NO:37) and GTGlacl XhoI-5′ (5′-CCGCTCGAGAGGGTGGTGAATGTGAAACCAGTAACGTT-3′) (SEQ IDNO:38). The resulting 1.1 kb PCR fragment was cloned into pCR-BluntII-TOPO to create pCR-Blunt II-TOPO-Lacl(E-X). The relA upstreamhomology region from the X 3761 genome was amplified using the primerpairs RelA N-HindIIISacI-5′(5′-CCCAAGCTTGAGCTCGAGGGCGTTCCGGCGCTGGTAGAA-3′) (SEQ ID NO:39) and RelAN-BglII-3′ (5′-GAAGATCTAAGGGACCAGGCCTACCGAAG-3′) (SEQ ID NO:40). Thefragment was cut with HindIII-BglII and ligated into plasmid pYA3700 atthe same restriction sites to generate plasmidpGEM3Z-pBADaraCT4ipIIIrelA-N. Plasmid pYA3700 was cut with XhoI-XbaI andligated into pCR-Blunt II-TOPO-Lacl(E-X) to generate the plasmidpCR-Blunt II-TOPO-LaclpBADaraC. This plasmid was cut with EcoRI, bluntedwith Mungbean nuclease and then cut with HindIII. PlasmidpGEM3Z-pBADaraCT4ipIIIrelA-N was cut with XbaI, blunted with Mungbeannuclease and then cut with HindIII. These two fragments were ligated toform the plasmid pCR-Blunt II-TOPO-Lacl(GTG)pBADaraC-relAN. The relAdownstream homology region was amplified from χ3761 genome using theprimer pairs RelA C-EcoRI-5′ (5′-CGGAATTCACCCCAGACAGTAATCATGTAGCGGCT-3′)(SEQ ID NO:41) and RelA C-KpnI-3′(5′-CGGGTACCCCAGATATTTTCCAGATCTTCAC-3′) (SEQ ID NO:42). The fragment wasligated with the pCR-Blunt II-TOPO-Lacl(GTG)pBADaraC-relAN, cut withXbaI and blunted with Mungbean nuclease, to generate plasmid pYA3782.The relA::araC P_(BAD) lacl TT cassette was cut with KpnI-SacI andcloned into pRE112 to generate the suicide plasmid pYA3784 harboring theGTG-lacl. The ATG-lacl was amplified using primers pairs, lacl EcoRI-3′(5′-GGAATTCTCACTGCCCGCTTTCCAGTCGGG-3′) (SEQ ID NO:43) and XhoI SD*-ATGlacl-5′ (5′-CCGCTCGAGAGGATGGTGAATATGAAACCAGTAACGTT-3′) (SEQ ID NO:44)and cloned into pCR-Blunt-II-Topo vector. The codon optimization ofATG-lacl was done by the PCR method. Briefly, 22 pairs of overlappingprimers covering 15 non-optomized codons used in the lacl nucleic acidsequence were PCR amplified. The overlapping PCR products were used astemplate to be amplified again to get codon optimized ATG-lacl. The 15codons are 35th CGG to CGT, 49th CCC to CCG, 101th CGA to CGT, 155th CCCto CCG, 168th CGA to CGT, 213th ATA to ATC, 216th CGG to CGT, 239th CCCto CCG, 272th GGA to GGT, 320th CCC to CCG, 326th AGA to CGT, 332th CCCto CCG, 339th CCC to CCG, 351th CGA to CGT and 355th CGA to CGT. Thecodon optimized ATG-lacl was also cloned into the pCR-Blunt-II-Topoplasmid. Then the similar strategies were used to generate suicideplasmid pYA3789 with the ATG-lacl and suicide plasmid pYA4064 with codonoptimized lacl.

Construction of Expression Plasmid pYA4088:

Plasmid pYA3494 carries amino acids 3-257 of native pspA Rx1 fused tothe first 23 amino acids of bla [23]. Nine codons that are rare inSalmonella were converted to highly used codons without changing theamino acid sequence to create plasmid pYA3635 using PCR methods [58].The 9 codons are 2nd CCC to CCG, 57th CTA to CTG, 77th CTA to CTG, 95thATA to ATC, 113th CGA to CGT, 144th CTA to CTG, 185th AGA to CGT, 186thCTA to CTG, 221 st CTA to CTG. Two additional codons, 23rd GCG to GCTand 124th GCT to GCG were also changed to keep the GC content the same.Generally, the overlapping primers covering the 9 non-optimized codonsused in the pspA Rx1 nucleic acid sequence were PCR amplified. Theoverlapping PCR products were used as template to be amplified again toget the codon optimized pspA Rx1. The final PCR product was cloned intopYA3493 to generate pYA3635. Using pYA3635 as the starting material, theoptimized pspA sequence was extended an additional 28 amino acids toinclude a recently identified B cell epitope (S. Hollingshead, personalcommunication). The extended codon-optimized pspA Rx-1 nucleic acidsequence was constructed in 3 steps. First, the optimized pspA sequencewas amplified using primer set PspA Rx1 forward(5′-TCTCCGGTAGCCAGTCAGTCTAAAGCTGAG-3′) (SEQ ID NO:45) and PspA RX1-a1(5′-CTAATTCAGCTTTTTTAGCAGCAATAGTTTTCTCTAAACCTTCTTTAAAGTAGTCTTCTACATTATTGTTTTCTTC-3′) (SEQ ID NO:46). The resulting 820-bp PCR fragmentwas used as template in a second PCR reaction using primer set PspA Rx1forward (5′-TCTCCGGTAGCCAGTCAGTCTAAAGCTGAG-3′) (SEQ ID NO:47) and PspARx1-a2 (5′-TGCTTTCTTAAGGTCAGCTTCAGTTTTTTCTAATTCAGCTTTTTTAGCAGCAATAGTTTTCTC-3′) (SEQ ID NO:48) PspA Rx1-EcoRI-s. The resulting 849-bp PCRproduct was used as template for a third amplification with the primerset PspA Rx1-EcoRI-s (5′-GGAATTCTCTCCGGTAGCCAGTCAGTCT-3′) (SEQ ID NO:49)and PspA Rx1-HindIII-a (5′-TTCAAGCTTATTATGCTTTCTTAAGGTCAGCTTC-3′) (SEQID NO:50). The 869-bp PCR product from that reaction was cloned intoplasmid pYA3493 [23] using EcoRI-HindIII restriction sites to generatepYA4088. The sequence was verified by sequencing and enzyme digestion.

Construction of Plasmid pYA4090:

Plasmid pYA3552 comprises the gfp3 nucleic acid sequence, which is akind gift from Dr. Ho-Young Kang. Plasmid pYA4090 was constructed by PCRamplification of the 740-bp gfp3 nucleic acid sequence using plasmidpYA3552 as template with the primer set GFP-EcoRI-s(5′-GGGAATTCCGATGAGTAAAGGAGAAGAACTTTTC-3′) (SEQ ID NO:51) andGFP-HindIII-a (5′-CGGTGCAAGCTTATTATTTGTATAGTTCATCCATG-3′) (SEQ ID NO:52)and then cloned into pYA3342 using EcoRI-HindIII.

Construction of Plasmid pYA3438:

The plasmid containing the 1.5 kb pabB nucleic acid homology was clonedinto the XbaI-BamHI site of pMEG375. The pabB nucleic acid sequence had106 bp deleted between the two internal EcoRV sites.

Construction of plasmid pYA3599: The suicide plasmid pYA3599 was used todelete the araBAD operon. A 360 bp fragment of the 3′ end of araD wasgenerated by PCR using primers araD-BamHI (5′-CGGGATCCTGGTAGGGAACGAC-3′;add BamHI underlined) (SEQ ID NO:53) and araD-NcoI(5′-GATGCCATGGTTTAAACTATATTCAGCAAATGCG-3′; add NcoI underlined) (SEQ IDNO:54), and a 500 bp fragment of 5′ end of the araB nucleic acidsequence was nucleic acid generated by PCR using primers araC-NcoI(5′-GATGCCATGGTCTGTTTCCTCGTCTTACTCCATCC-3′; add NcoI underlined) (SEQ IDNO:55) and araC-SphI (5′-ACATGCATGCGGACGATCGATAA-3′; add SphIunderlined) (SEQ ID NO:56). These two fragments were cloned into theBamHI and SphI site of pMEG-375 to result in the suicide vector pYA3599.

Construction of χ9097:

The strain χ8060 is from Megan Health, Inc. and harbors a pabA 1516mutation. The χ8442 strain was constructed by conjugation of χ7213,harboring plasmid pYA3599, with χ8060. The χ8914 strain was constructedby conjugation of χ7213, harboring plasmid pMEG-443, with χ8060. The □χ8767 was constructed by conjugation of χ7213 harboring plasmid pYA3599.The P22 lysate was made on the single cross over by conjugation χ7213,harboring plasmid pYA3599, with □ χ8767 according to Kang's method [59].The ΔaraBAD23 mutation was introduced into strain χ8914 by P22transduction from (χ8767:pYA3599) to generate strain χ9097. The mutationwas verified by PCR and formation of a white colony phenotype onMacConkey agar supplemented with 1% arabinose. Minimal agar with/withoutpABA was used to detect the phenotype associated with the pabA pabBmutations. The presence of the asdA mutation in Salmonella was confirmedby its dependence on DAP for growth [21]. The presence of the 3.3 kbdeletion-insertion of relA was confirmed by PCR with primer set RelAN-HindIIISacI-5′ (5′-CCCAAGCTTGAGCTCGAGGGCGTTCCGGCGCTGGTAGAA-3′) (SEQ IDNO:57) and RelA C-KpnI-3′ (5′-CGGGTACCCCAGATATTTTCCAGATCTTCAC-3′) (SEQID NO:58) and western-blot using anti-Lacl antiserum as described below.Lipopolysaccharide (LPS) profiles of Salmonella strains were examined asdescribed [60].

Construction of Vectors and Strains:

Similar strategies to those described above were used to constructpYA3789 and pYA4064 to generate the ATG-lacl mutation DrelA197::araCP_(BAD) lacl TT and the codon optimized ATG-lacl mutation ΔrelA198::araCP_(BAD) lacl TT, respectively. Plasmid pYA3342 is an Asd+ expressionvector with promoter P_(trc) [23]. Plasmid pYA4090 is a pYA3342derivative that codes for gfp3 expression from the P_(trc) promoter.Details for the construction of plasmid pYA4090 are described herein.Plasmid pYA3493 is a pYA3342 derivative that encodes the first 23 aminoacids of β-lactamase [23]. Plasmid pYA4088, derived from pYA3493,carries a cloned fragment of the S. pneumoniae pspA nucleic acidsequence, encoding aa 3-285, that has been codon-optimized forexpression in Salmonella, and fused to the nucleic acid sequenceencoding amino acids 1-23 of β-lactamase.

Construction and Phenotypic Characterization of S. typhimurium VaccineStrains:

The ΔrelA196::araC P_(BAD) lacl TT, ΔrelA197::araC P_(BAD) lacl TT andΔrelA198::araC P_(BAD) lacl TT mutations were introduced into the S.typhimurium strain χ3761 by allelic exchange using χ7213 harboring thesuicide vectors pYA3784, pYA3789 and pYA4064 to yield χ8990, χ9080 andχ9226, respectively, and into RASV strain χ9097 to generate χ9095, 9101and 9241. The presence of the 3.3 kb deletion-insertion was confirmed byPCR and western-blot as described below.

Western Blot Analysis:

Protein samples were prepared from equal numbers of cells, separated ona 12% SDS-PAGE gel, and transferred to a nitrocellulose membrane usingTrans-Blot SD Semi-Dry Transfer Cell (Bio Rad). Lacl, PspA and GroELwere detected using rabbit polyclonal anti-Lacl, anti-PspA andanti-GroEL primary antiserum, respectively, at 1:10,000 dilutions, and asecondary anti-rabbit alkaline phosphatase-conjugated antibody (Sigma,St Louis, Mo.) at 1:10,000 dilution. Bands were visualized usingNBT/BCIP (Sigma,). The bands were scanned and densitometry was measuredusing Quantity One software (Bio-Rad).

Growth Curves:

Standing overnight 37° C. cultures of RASV strains χ9095, χ9097, χ9101and χ9241, with and without plasmid pYA4088, were grown in LB or LB plusDAP, respectively, containing 0.2% arabinose. The culture was adjustedto the same OD with pre-warmed medium, and then diluted 1:100 intopre-warmed LB or LB-DAP broth with 0.2% arabinose. The optical densityat 600 nm (OD₆₀₀) was measured every 40 min. At the final time point,samples of each strain were taken and used for western blot analysiswith anti-Lacl and/or anti-PspA antisera.

Protein stability analysis: S. typhimurium strains χ8990, χ9080 andχ9226 were grown in 3XD medium containing 0.2% arabinose and E. colistrain XL1-Blue was grown in LB without arabinose. Standing overnightsof each strain were grown at 37° C., diluted 1:100 into fresh media andgrown with aeration to an OD₆₀₀ of 0.6. Cells were washed 2 times withfresh medium. Chloramphenicol was added to 50 μg/ml. Samples takenbefore adding chloramphenicol (pre 0), just after adding chloramphenicol(0), and at 1, 2, 4, 6, 8, 24 h were analyzed by western blot. Thesamples were normalized by cell number before loading onto the gel.

Flow Cytometry Analysis:

Standing overnight cultures of χ9095(pYA4090), χ9097(pYA4090), χ9101(pYA4090) and χ9241 (pYA4090) were grown at 37° C. in Nutrient Brothwithout arabinose. Then, 3×10⁵ CFU of each strain were added to 3 ml offresh medium containing 0%, 2%, 0.2%, 0.02% or 0.002% arabinose andgrown to an OD₆₀₀ of 0.4. The cultures were diluted 1:10 in PBS andsubjected to flow cytometry analysis using Cytomics FC500 (BeckmanCoulter, Inc., Fullerton, Calif., USA). The data were analized by CXPanalysis software (Beckman Coulter, Inc.)

Kinetics of Lacl Loss and Antigen Synthesis in Pre-Induced CulturesGrown without Arabinose:

Overnight cultures of strains χ9095, χ9097, χ9101 and χ9241 carryingeither plasmid pYA4088 or plasmid pYA4090 were grown in Nutrient brothwith or without 0.2% arabinose. Each culture was adjusted to OD₆₀₀=0.6and diluted 1:100 into the same pre-warmed medium at 37° C. Whencultures reached an OD₆₀₀ of 0.6, the cultures were washed once withnutrient broth without arabinose and diluted 1:100 (for plasmid pYA4090)or 1:10 (for plasmid pYA4088) into pre-warmed nutrient broth withoutarabinose and grown to an OD₆₀₀=0.6. The cultures were diluted intofresh media and the process was repeated twice (for pYA4090 cultures) orthree times more (for pYA4088 cultures). Samples were taken at the endof each growth cycle. Samples of strains χ9095(pYA4088), χ9097(pYA4088),χ9101 (pYA4088) and χ9241(pYA4088) were normalized according to cellnumber and analyzed by western blot. Bands were scanned and densitometrywas measured using Quantity One software (Bio-Rad, Hercules, Calif.).Samples of strains χ9095(pYA4090), χ9097(pYA4090), χ9101 (pYA4090) andχ9241(pYA4090) were analyzed by flow cytometry.

Tests of Immunogenicity and Protection in Mice:

Strains were grown in LB medium supplemented with 0.2% arabinose to anOD₆₀₀ of 0.8, sedimented by room temperature centrifugation at 6,000×gfor 15 minutes and resuspended in phosphate-buffered saline containinggelatin (BSG) [24]. Groups of female BALB/c mice were orally immunizedwith 10⁹ CFU of the RASV. Food and water was removed 4 h beforeinoculation and restored 30 min after inoculation. The inoculum wasdiluted for titer determination on LB agar plates. Mice were bled at 0,2, 4, 6, and 8 weeks. Anti-LPS and anti-PspA Rx1 serum IgG was evaluatedby ELISA. Mice were challenged by intraperitoneal injection with 250LD₅₀ of virulent S. pneumoniae WU2 eight weeks after immunization. Themice were observed daily for 21 days after challenge. All animalprotocols were approved by ASU IACUC and complied with rules andregulations by American Association for Accreditation of LaboratoryAnimal Care.

ELISA:

rPspA Rx1 protein was purified as described by Kang et al. [23]. S.typhimurium LPS was obtained from Sigma. The procedure for ELISA hasbeen described [23]. Briefly, polystyrene 96-well flat-bottom microtiterplates (Nunc, Roskilde, Denmark) were coated with 100 ng/well of eitherS. typhimurium LPS or rPspA Rx1 in 100 ml sodium carbonate-bicarbonatecoating buffer (pH 9.6). The sera were serially diluted in two-foldsteps for detection of IgG. A 100 ml of diluted sample was added totriplicate wells. Plates were treated with biotinylated goat anti-mouseIgG (Southern Biotechnology Inc., Birmingham) and then alkalinephosphatase-labeled streptavidin (Southern Biotechnology Inc.,Birmingham). After adding p-nitrophenylphosphate substrate solution indiethanolamine buffer (pH 9.8) (Sigma, St. Louis, Mo.), absorbance wasread at 405 nm.

Statistics:

Statistical analyses were performed by using the SPSS software package(SPSS, Chicago, Ill.). p values of 50.05 were considered significant.Antibody titers were expressed as means±standard error. The means wereevaluated with One-way Anova and the LSD tests were used for multiplecomparisons among groups.

Rationale for the Regulated Delayed Antigen Synthesis System

The P_(trc) promoter is commonly used for constitutive expression ofnucleic acid sequences encoding antigens [25, 26]. P_(trc) is a strongpromoter in vivo [27], constitutive under most environmental conditions,and is more transcriptionally active both anaerobically and aerobicallythan the nirB promoter [14]. Although the P_(trc) promoter has beenwidely used in bacterial expression vectors and in mammalian cellexpression systems [28-30], it has been reported that constitutiveantigen expression from the similar P_(tac) promoter can affect thecolonization capability of RASV [16]. Thus regulating antigen expressionfrom P_(trc) would be beneficial. The P_(trc) promoter can be repressedby Lacl. In Escherichia coli, the Lacl repressor is typically producedat approximately 8 copies per cell [31, 32] and regulates expression ofthe lactose metabolic nucleic acid sequences [33] by binding to the lacOoperator sequence, blocking RNA polymerase from binding to the lacpromoter. Generally, induction of expression from Lacl-repressedpromoters is accomplished by the addition of chemical agents, eitherlactose or IPTG which bind to Lacl causing an allosteric change in theprotein that leads to its release from lacO. However, this is not apractical method of induction for RASVs. Instead, the production of Laclwas regulated by placing it under the control of the regulatedarabinose-inducible promoter P_(BAD) (FIG. 19A). Transcription fromP_(BAD) can be regulated by varying the concentration of arabinose [34,35]. When the RASV is grown in culture and arabinose is added to thegrowth medium, Lacl is produced which represses transcription fromP_(trc). Once in the host, the transcription from P_(BAD) would ceasebecause free arabinose is very rarely encountered in animal tissues[36], and subsequently, no additional Lacl would be produced. Theconcentration of Lacl should then decrease by dilution as the RASV cellsdivide and antigen production would increase to levels high enough toinduce the desired immune response.

Construction of Strains with High Expression of Lacl

Based on this concept, a tightly-regulated araC P_(BAD) lacl TT cassettewas integrated into the chromosome in the relA nucleic acid sequence(FIG. 19B), nucleic acid sequencing defining the relA 196 allele instrain, χ8990. relA was chosen as the integration site because the relAmutation is not attenuating nor does it have an effect on colonization[37, 38]. The native lacl nucleic acid sequence has a GTG start codonand an AGGG Shine-Dalgarno sequence, leading to expression of only 5-10molecules each generation [31] and the functional form of Lacl repressoris a tetramer [32]. Plasmids with a ColE1 (pBR) replicon are present at20-30 copies per cell. Thus, it was estimated that at least 80-120copies of Lacl are needed to repress the operator sequences in theexpression plasmid to achieve adequate repression. Therefore, inaddition to strain χ8990, which encodes the native lacl sequence(GTG-lacl), the start codon of lacl was modified from GTG to ATG, andthe SD sequence was modified from AGGG to the canonical ribosome bindingsite sequence, AGGA, resulting in the relA197 allele in strain χ9080.This modification increased Lacl levels about 2 fold (FIG. 19C). Toenhance expression further, the codons of lacl were optimized accordingto the codon usage for highly expressed nucleic acid sequences inSalmonella, yielding the relA198 allele in strain χ9226. Thismodification increased Lacl levels approximately 4-8 fold over relA196(FIG. 19C). The expression levels of Lacl for GTG-lacl, ATG-lacl, andcodon optimized-lacl were proportional to the arabinose concentration(FIG. 19C). It is anticipated that different antigens may requiredifferent levels of repression, so these constructs provide theflexibility needed to produce different amounts of repressor to meetvaried requirements.

High Expression of Lacl does not Affect Growth.

The Salmonella chromosome does not encode the lac operon. Consequently,the effect of Lacl production or overproduction on growth wasinvestigated, because this might translate to a reduction inimmunogenicity. The growth of all three of the above strains wasevaluated in LB broth with or without 0.2% arabinose, and compared to astrain that does not produce Lacl. All four strains had similar growthrates, including strain χ9241 (relA198), which produces the most Lacl(FIG. 20). Although one of the Lacl-producing strains, χ9101 (relA197),grew better than the other strains, there were no large differences indoubling times. Growth at lower concentrations of arabinose gave similarresults, but 2% arabinose led to reduced growth of all theLacl-producing strains.

Codon Optimized Lacl Provides the Highest Repression

To evaluate the relationship between antigen synthesis and arabinoseconcentration, plasmid pYA4090, which encodes the gfp3 nucleic acidsequence under transcriptional control of P_(trc), was introduced intoS. typhimurium strains χ9097, χ9095, χ9101 and χ9241. Transformants weregrown in LB at varying arabinose concentrations and subjected to FACSanalysis. The P_(BAD) promoter is subject to autocatalytic regulation,and therefore we were able to evaluate the fraction of cells expressingGFP as a measure of induction [39]. As expected, there was no effect ofarabinose on gfp3 nucleic acid expression in strain χ9097(pYA4090),which does not encode lacl (FIG. 21A). When strains χ9095(pYA4090),χ9101(pYA4090) and χ9241(pYA4090) were grown without arabinose, nearlyall the cells expressed GFP. No decrease in the number of GFP-positivecells was observed when 0.002% arabinose was included in the growthmedium but repression was evident at 0.02% arabinose for all strains. Asthe arabinose concentration was increased, the number of GFP positivecells dropped substantially for all strains with arabinose-regulatedexpression of lacl. The greatest level of repression was seen in strainχ9241 (relA198), with only 7.6% GFP positive cells in the presence of 2%arabinose. These results are consistent with the expectation thatantigen synthesis should be inversely proportional to arabinoseconcentration and inversely proportional to Lacl synthesis (FIG. 21A).Although the lacl constructs in χ9095 (relA196) and χ9101 (relA197)produced different amounts of Lacl (FIG. 19), there was no difference ingfp nucleic acid expression between the two strains.

Lacl is a Stable Protein

Because Lacl is not normally synthesized in S. typhimurium and in E.coli it is only expressed at low levels and because of the central roleLacl plays in the system, the stability of Lacl was investigated inthese strains, as that could have an impact on the timing of antigensynthesis in vivo. Chloramphenicol was added to mid-exponential phasecultures of Salmonella strains χ8990, χ9080 and χ9226 and E. coli strainXL1-Blue. The stability of Lacl was similar for all strains (FIG. 22).The amount of Lacl declined by 50% over the first 2-4 hours in allstrains, after which the amount declined very slowly, reachingapproximately 20% of the starting levels by 24 hours for the S.typhimurium strains and 40% for the E. coli strain. These resultsindicate that Lacl stability is essentially the same in S. typhimuriumand E. coli, and that the protein is relatively stable. Thus it isexpected that the concentration of Lacl in these strains, in the absenceof arabinose, will decrease primarily due to dilution as a result ofcell division.

Time Course for the Induction of GFP Synthesis.

To evaluate the kinetics of the induction of antigen synthesis aftergrowth in arabinose, the GFP-producing strains were grown in nutrientbroth with 0.2% arabinose. Nutrient broth was chosen as the growthmedium because it is derived from animal tissue and should mimic the lowarabinose conditions found in host tissues better than LB broth. Thearabinose-grown cells were diluted 1:100 into fresh nutrient brothwithout arabinose and grown to an OD₆₀₀ of 0.6. The cells were dilutedand grown twice more in the same way. Each round of growth representedapproximately 4.3 cell divisions, for a total of 13.8 nucleic acidsequencerations of growth in the absence of arabinose. Samples weretaken at the end of each growth cycle and analyzed by FACS (FIG. 21B).The results indicated that although some strains were not fullyrepressed by growth in arabinose, the kinetics of induction was similarin all strains leading to nearly full induction for synthesis of GFP by9.2 nucleic acid sequencerations of growth.

Time Course for the Induction of PspA Antigen Synthesis

As shown above, the system worked as expected using gfp3 as a model. Thesystem was next evaluated in the context of a vaccine with a clinicallyrelevant antigen, the S. pneumoniae PspA Rx1 protein that has been shownto be a potent and protective immunogen [23]. Plasmid pYA4088 wasintroduced into the strains and their growth rates in LB broth werecompared. All of the Lacl-producing strains had a growth advantage overstrain χ9097(pYA4088) in the presence of 0.2% arabinose (FIG. 20B),indicating that repressing the expression of the nucleic acid sequenceencoding antigen results in faster growth. The Lacl-producing strainsalso grew faster than χ9097(pYA4088) in the absence of added arabinose.This may be due to trace amounts of arabinose present in the yeastextract used to prepare the LB broth medium, approximately 0.0034% (K.Ameiss, personal communication). Although the amount of Lacl producedunder these conditions was undetectable by western blot (FIG. 19C),there may have been sufficient Lacl present to account for the observedgrowth advantage.

Next Lacl and PspA synthesis were directly evaluated in cells grown in0.2% arabinose. This concentration was chosen because there was only asmall difference in repression levels between 2% and 0.2% arabinose,(FIG. 21A) and the addition of 2% arabinose resulted in a growth ratereduction. The amount of PspA synthesis was inversely correlated withLacl synthesis, as expected (FIG. 21C). PspA synthesis levels in strainχ9241(pYA4088), which produced the most Lacl, were approximately 8-foldless than the χ9097(pYA4088) control. The kinetics of induction wasevaluated as described above for the GFP-producing strains and it wasfound that all of the strains produced the same amount of PspA asχ9097(pYA4088) after 9.2 nucleic acid sequencerations of growth,indicating full derepression of P_(trc) (FIG. 21C). For most strains,the maximum PspA synthesis was achieved by 6.9 nucleic acid generations.

Regulated Delayed Expression Provides Better Protection thanConstitutive Expression

Strains χ9097(pYA4088), χ9095(pYA4088), χ9101 (pYA4088) andχ9241(pYA4088) were then evaluated for immunogenicity in mice in twoseparate experiments. The results of both experiments were similar, sothe results were pooled. After a single dose, all immunized micedeveloped high titers against S. typhimurium LPS (FIG. 23A). Comparedwith vector controls, the strains synthesizing PspA elicited lower LPStiters, although the synthesis of Lacl abrogated this effect somewhat.While all the RASV strains carrying pYA4088 induced a strong anti-PspAserum IgG response (FIG. 23B), those strains, χ9095, χ9101 and χ9241that produced Lacl induced significantly higher anti-PspA titers thanstrain χ9097(pYA4088) (p<0.05). Strain χ9241(pYA4088) vaccinatesproduced higher anti-PspA antibodies than the other strains at 6 and 8weeks (p<0.05). Overall, the serum antibody response was roughlyproportional to the amount of Lacl produced by each strain. No anti-PspAantibody response was detected in mice vaccinated with the vector onlycontrols.

When vaccinated mice were challenged with virulent S. pneumoniae WU2,all groups that received PspA-producing strains were protected (p<0.01;FIG. 24). Among the protected groups, mice vaccinated with strain χ9101(pYA4088) showed significantly higher protection than mice vaccinatedwith the other strains (p<0.05). The remaining vaccinated groups werenot significantly different from each other (p>0.05). Interestingly,χ9241(pYA4088), the strain that induced the highest anti-PspA antibodytiter (FIG. 23B), provided the poorest protection. These resultsillustrate the need to optimize the amount of Lacl and antigen to attainan optimal immune response and indicate that protection may not beclosely correlated with induced antibody titer levels.

Discussion for Example 1

A regulated delayed expression system has been developed to minimize thenegative effects of antigen expression on the host strain and to enhanceimmunity. An araC P_(BAD) lacl TT cassette was engineered to synthesizedifferent levels of Lacl when grown in the presence of arabinose (FIG.19C). The amount of Lacl produced by the strains was proportional to theamount of arabinose present in the medium, up to 2%, the maximumconcentration tested (FIG. 19C). These results differ from what has beenobserved in E. coli where protein synthesis from P_(BAD) reached amaximum at 0.2% arabinose [34]. There are several possible reasons forthis difference. First, the source of the araC P_(BAD)promoter/activator cassette was E. coli K-12, while in the previousstudies, the promoter was derived from E. coli B/r. Tighter regulationhas been observed with a K-12 cassette than with a B/r cassette.Additionally, the previous studies were performed using plasmid copiesof P_(BAD), while herein the P_(BAD) was chromosomally integrated.Moreover, differences in regulation have been observed for a number ofpromoters when they are present in multiple copies (Kenneth Roland,personal communication). Finally, differences in the arabinose-transportsystem between E. coli and Salmonella may have also played a role.Salmonella only has one L-arabinose transport system encoded by araE[40, 41], which has a low affinity for arabinose, while E. coli has botharaE and the high affinity transport system encoded by araFGH [40, 42].Therefore, in Salmonella, higher concentrations of arabinose are likelyto be required for full P_(BAD) promoter induction than in E. coli [40].

Although 2% arabinose can induce the maximum Lacl synthesis, repressionof antigen synthesis was not complete (FIG. 21). One reason for thisproblem is undoubtedly the fact that the P_(trc) promoter is present ona multicopy plasmid, while Lacl is specified by a single nucleic acidsequence on the chromosome. Another reason may be that the bindingaffinity of the lactose operator sequence in P_(trc) is 10-fold lessthan the ideal one [43]. An additional consideration is that in thenative E. coli lac operon, there are 3 adjacent operator sequences,facilitating cooperative binding interactions between three Lacltetratmers [44], while there is only one operator in our plasmid[45-47]. Consistent with this hypothesis is the fact that only in strainχ9241, which produced 3-4 times the amount of Lacl as the other strains,was antigen expression nearly shut off completely. Thus it is likelypossible to improve the efficiency of antigen repression by modifyinglacO for tighter repressor binding. In addition, one can also modify thesystem by adjusting the amount of arabinose in the growth medium,thereby varying the amount of Lacl in the cell.

In this example, three strains were developed with the idea thatdifferent antigens may require more or less Lacl to achieve anappropriate balance between the health of the RASV and the optimalantigen expression required for induction of protective immuneresponses. Antigen expression was reduced in vitro, which led to afaster growth rate by the RASV (FIG. 20). While all the RASV strainsdescribed in this example grew faster than the control strain thatconstitutively expressed PspA, the results from each strain weredifferent with respect to the serum immune response and protectiveimmunity (FIG. 23, FIG. 24). It was anticipated that strain χ9241 withthe relA198 allele would elicit the highest antibody titers and providethe best protection. This strain did, in fact, yield the highestanti-PspA serum IgG titers (FIG. 23), but it did the poorest job ofproviding protection against S. pneumoniae challenge (FIG. 24). This maybe a reflection of differences in the amount of antigen produced by eachstrain and the timing of the induction of antigen synthesis or thatother factors such as cellular immunity might be more important thanantibodies for conferring protective immunity.

In conclusion, a regulated delayed antigen expression system has beendeveloped. This system reduces the negative effects of antigenexpression during in vitro growth thereby improving the overall healthof the vaccine strain, while allowing for maximum antigen expression inhost tissues. This technology should be particularly useful for inducingimmune responses to antigens that are toxic to the vaccine strainsynthesizing them.

REFERENCES FOR EXAMPLE 1

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Sizemore, Construction and    evaluation of a delta cya delta crp Salmonella typhimurium strain    expressing avian pathogenic Escherichia coli 078 LPS as a vaccine to    prevent airsacculitis in chickens. Avian Dis, 1999. 43(3): p.    429-41.-   49. Curtiss, R., III., Colonization Control of Human Bacterial    Enteropathogens in Poultry, J. Bailey, Cox N A, Stern N J.    Meinersmann R J, Editor. 1991, Academic Press, New York p. 169-198.-   50. Curtiss, R., III. and S. A. Tinge, Regulated antigen delivery    system (RADS), in U.S. Pat. No. 6,780,405. 2004: US.-   51. Sambrook, J. and D. W. Russell, Molecular cloning: a laboratory    manual. 3rd ed. 2001, Cold Spring Harbor, N.Y.: Cold Spring Harbor    Laboratory Press.-   52. O'Callaghan, D. and A. Charbit, High efficiency transformation    of Salmonella typhimurium and Salmonella typhi by electroporation.    Mol Gen Nucleic acid sequencet, 1990. 223(1): p. 156-8.-   53. Schmieger, H. and H. 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Example 2: Regulated Attenuation

Attenuation of Salmonella vaccine vectors should decrease, if noteliminate, undesirable disease symptoms, but the nutritional status andhealth of the population to be vaccinated should be considered. Theattenuation should be (i) an inherent property of the vaccine and notdepend on fully functional host defenses and immune responses, (ii) notbe reversible by diet or by host or microbial modification of dietconstituents, and (iii) not permit development of a persistent carrierstate. The attenuated vaccine should be sufficiently invasive andpersistent to stimulate both strong primary and lasting memory immuneresponses and should be designed to minimize unnecessary tissue damage.As even attenuated vaccines may cause disease in unlucky individuals,the vaccine should be susceptible to clinically useful antibiotics. Manymeans to attenuate Salmonella vaccines make them less able to toleratestresses encountered after oral administration including exposure toacid, bile, increasing osmolarity and iron, and decreasing O₂, and/orreduced invasion of the gut associated lymphoid tissue (GALT). The dosesof recombinant Salmonella vaccines to elicit maximal immune responsesare lower for intranasal immunization than they are for oralimmunization (1,2). This may be due, in part, to killing of orallyadministered vaccines by the acid stress of the stomach (3) quicklyfollowed by exposure to bile in the duodenum. We have determined thatthese two stresses in succession are more effective in causing bacterialcell death than the sum of killing by each stress alone. Salmonellapossesses a large constellation of genes that confer acid tolerance andresistance to acid stress (4,5) and inactivation of these genes or theirinability to be expressed by induction, reduces virulence (6). In thisregard, the regulatory proteins RpoS (7), Fur (8), PhoPQ (9) and OmpR(10, 11) are all necessary to confer resistance to acid stress and/orshock in S. typhimurium. Similarly, many genes are turned on in responseto exposure to bile and some of these gene products transiently repressinvasion while bacteria reside in the intestinal lumen (12-14).

It is important to have mutations contributing to attenuation or otherbeneficial vaccine attributes that do not impair the abilities of thevaccine to adjust to and/or withstand a diversity of stressesencountered at any location within the gastrointestinal tract ifadministered orally or in the respiratory tract if administeredintranasally. Likewise, the vaccine strain should have wild-typeabilities not compromised by attenuating or other mutations to penetratethrough mucin, to attach to cells in the mucosal epithelium and beinvasive into those cells. To achieve these objectives, means have beendeveloped herein to achieve regulated delayed attenuation in vivo suchthat the vaccine at the time of immunization exhibits almost the sameabilities as a fully virulent wild-type strain to contend with stressesand successfully reach effector lymphoid tissues before display ofattenuation to preclude onset of any disease symptoms. The meansdescribed herein confer high-level attenuation and superiorimmunogenicity compared to traditional mutationally attenuated strains.

Materials and Methods for Example 2

Bacterial Strains, Media and Bacterial Growth:

All strains for testing in mice are derived from the highly virulent S.typhimurium strain UK-1 (15). All bacterial strains for this example arelisted above in Table 1. LB broth and agar (16) are used as complexmedia for propagation and plating of bacteria. Nutrient broth and agar(Difco), which are devoid of arabinose and mannose, and minimal saltsmedium and agar (17) were also used. Some studies were done withbacterial strains grown in tissue culture medium to simulateenvironments to be encountered in vivo. MacConkey agar with 0.5% lactose(Lac), 0.2 or 0.5% arabinose (Ara) or 0.5% maltose (Mal) were used toindicate fermentation of sugars and enumerate bacteria from mice. CASplates (Schwyn B, Neilands J B, 1987. Universal chemical assay for thedetection and determination of siderophores. Anal Biochem. 1987 January;160(1):47-56), which were used to determine siderophore production, weremade by addition of chrome azurol S mixed with Fe⁺³ andhexadecyltrimethyl ammonium bromide (HDTMA) to MOPS basal agar. X-Pplates to detect phosphatase activity were made by addition of 50 mg/mlof 5-bromo-4-chloro-3-indolyl-phosphate (BCIP or XP) to Nutrient agar.Kornberg agar medium plates were prepared as a glycogen indicator agar(18-20). Selenite broth, with or without supplements, was used forenrichment of Salmonella from tissues, although later resultsdemonstrated that enrichment with tetrathionate broth gave betterresults when vaccine strains had multiple mutations. Bacterial growthwas monitored spectrophotometrically and by plating for colony counts.

Molecular and Genetic Procedures:

Methods for DNA isolation, restriction enzyme digestion, DNA cloning anduse of PCR for construction and verification of vectors are standard(21). DNA sequence analysis was performed in the DNA Sequence Laboratoryin the School of Life Sciences at ASU. All oligonucleotide and/or genesegment syntheses were done commercially. Overlapping PCR amplificationwith primers designed for specific modifications was used to optimizecodons for translational efficiency in Salmonella or to alter promoter,ribosome binding/Shine-Dalgarno (SD) and start codon sequences.Conjugational transfer of suicide vectors for generation of unmarkeddeletion and deletion-insertion mutations was performed by standardmethods (22, 23) using the suicide vector donor strain χ7213 (Table 1).Since live vaccine strains cannot display resistance to antibiotics,means were used to generate defined deletion mutations using suicidevector technologies that did not use drug-resistance markers or leavemolecular scars. Subsequently, these unmarked defined deletion mutationswith and without specific insertions were introduced into strains usingP22HT int (24, 25) transduction of suicide vectors integrated into thedeletion or deletion-insertion mutation followed by selection forsucrose resistance as described (26). Whenever insertion of a regulatorysequence might adversely effect expression of an adjoining gene, atranscription terminator (TT) was included to prevent such consequences.Strong TTs from bacteriophages were generally used. Plasmid constructswere evaluated by DNA sequencing, ability to complement various S.typhimurium mutant strains (Table 1) and for ability to specifysynthesis of proteins using gel electrophoresis and western blotanalyses. His- or GST-tagged proteins have been produced and used toobtain anti-protein rabbit antisera for western blot analyses.

Strain Characterizations:

Exquisite care was taken in strain construction and complete biochemicaland genetic characterizations were performed after every step in strainconstruction. This includes running an LPS gel to make sure roughvariants were not selected. Comparative growth analyses were conductedsince the objective is to have single and multiple mutant strains growat similar rates and to the same density as the wild-type parentalstrains when grown under permissive conditions. Vaccine strain stabilitywas also evaluated, due to possible recombinational and/or mutationalevents as described below. Strains are also evaluated for biochemicaland metabolic attributes, sensitivity to antibiotics and drugs,serological properties and resistance compared to wild-type parentalstrains to stresses associated with exposure to acid and bile.

Cell Biology:

The ability of various constructed Salmonella strains to attach to,invade into and survive in various murine and human epithelial and/ormacrophage cell lines are quantitated by well established methods (27,28) that are used routinely.

Animal Experimentation:

BALB/c and C57BL/6 female mice, six to eight weeks of age, were used formost experiments. Mice are held in quarantine one-week before use inexperiments. They are deprived of food and water 6 h before oralimmunization. No bicarbonate is administered. Food and water arereturned 30 min after immunization. Candidate vaccine strains arequantitatively enumerated in various tissues as a function of time afterinoculation (29, 30). The inoculation procedures are the same as in theimmunization studies. All animals are housed in BL2 containment withfilter bonnet covered cages. If high immunogenicity is observed ininitial tests after primary immunization, subsequent studies are done todetermine the lowest level of vaccine inocula to induce a significantprotective immune response to oral or intraperitoneal challenge with thewild-type S. typhimurium UK-1 parental strain χ3761.

Construction of Deletion-Insertion Mutations to Achieve RegulatedDelayed Attenuation.

Four means are described to permit a regulated delayed attenuationphenotype so that vaccine strains at the time of immunization exhibitnearly wild-type attributes for survival and colonization of lymphoidtissues and after five to ten cell divisions become avirulent. Thesemeans to achieve regulated delayed attenuation rely on using an araCP_(BAD) activator-promoter that is more tightly regulated by arabinose(31) than the original sequence from the E. coli B/r strain (32). Thepromoter was deleted, including all sequences that interact withactivator or repressor proteins, for the fur, phoPQ, rpoS and crpnucleic acid sequences and substituted by insertion of the improved araCP_(BAD) cassette (31) to yield Salmonella strains with theΔP_(fur33)::TT araC P_(BAD) fur, ΔP_(phoPQ107)::TT araC P_(BAD) phoPQ,ΔP_(rpoS183)::TT araC P_(BAD) rpoS and ΔP_(crp527)::TT araC P_(BAD) crpdeletion-insertion mutations (P stands for promoter and TT stands fortranscription terminator). The suicide vectors used to generate thesefour deletion-insertion mutations are depicted in FIG. 25 and are listedin Table 2. A strong phage-derived TT was included at the 3′ end of thearaC nucleic acid sequence in all these constructions since itstranscription in the presence of arabinose could often lead to alteredover expression of downstream adjacent nucleic acid sequences with thesame transcriptional orientation as the araC nucleic acid sequence or todiminished expression when the downstream adjacent nucleic acid sequenceis in opposite orientation resulting in synthesis of anti-sense mRNAfrom P_(araC).

Phenotypic Characterization of Mutant Strains.

Growth of these strains in the presence of arabinose leads totranscription of the fur, phoPQ, rpoS and/or crp nucleic acid sequencesbut nucleic acid sequence expression ceases in the absence of arabinose.These activities can be readily observed by appropriate tests. Thusχ9021 with the ΔP_(crp527)::TT araC P_(BAD) crp deletion-insertionmutation can only ferment maltose when grown in the presence ofarabinose and not in the absence of arabinose as revealed by streakingcultures on MacConkey maltose agar without and with 0.2 percentarabinose (FIG. 26A). Similarly, 8848 with the ΔP_(fur33)::TT araCP_(BAD) fur and χ9107 with ΔP_(fur33)::TT araC P_(BAD) fur andΔP_(crp527)::TT araC P_(BAD) crp mutations reveal siderophore productionwhen streaked on CAS plates without arabinose and no siderophoreproduction when grown in the presence of arabinose (FIG. 26B). χ8918with the ΔP_(phoPQ107)::TT araC P_(BAD) phoPQ and χ9108 with theΔP_(phoPQ107)::TT araC P_(BAD) phoPQ and ΔP_(crp527)::TT araC P_(BAD)crp mutations when streaked on X-P plates without and with 0.2 percentarabinose reveal acid phosphatase activity due to expression of thePhoP-activated phoN nucleic acid sequence only when grown in thepresence of arabinose (FIG. 26C). χ8956 with the ΔP_(rpoS183)::TT araCP_(BAD) rpoS and 9064 with the ΔP_(rpoS183)::TT araC P_(BAD) rpoS andΔP_(crp527)::TT araC P_(BAD) crp mutations reveal glycogen accumulationwhen streaked on glycogen-indicator agar with 0.2 percent arabinose andsprayed with iodine indicator solution (FIG. 26D). The presence orabsence of RpoS in these strains can also be revealed by adding hydrogenperoxide to cultures to detect the activity of the RpoS-dependantcatalase, KatE, (33-36) when arabinose is present during strain growth.Since Crp positively enhances transcription from P_(BAD), the inclusionof the ΔP_(crp527)::TT araC P_(BAD) crp mutation with other araC P_(BAD)regulated nucleic acid sequences causes a tighter cessation oftranscription in the absence of arabinose. This is seen by closeexamination of the photographs in FIG. 26. For this reason, theΔP_(crp527)::TT araC P_(BAD) crp mutation is included in all vaccinestrains when using araC P_(BAD) regulation of virulence nucleic acidsequences.

Stability of Crp, Fur, RpoS and PhoP Proteins and their Decline DuringGrowth in the Absence of Arabinose.

Growth of strains with araC P_(BAD) regulated nucleic acid sequences inthe presence of arabinose results in acid production that can causecessation of growth. We have therefore included the ΔaraBAD23 mutation(FIG. 27) that prevents use of arabinose. Inclusion of this mutationalso prevents breakdown of arabinose retained in the cell cytoplasm atthe time of oral immunization and inclusion of the ΔaraE25 mutation(FIG. 27), which enhances retention of arabinose further delayscessation in expression of araC P_(BAD) regulated nucleic acid sequencesfor an additional cell division or so. The suicide vectors forintroducing the ΔaraBAD23 and ΔaraE25 mutations are listed in Table 2.

The stability of virulence gene products in strains with each of thearaC P_(BAD) regulated virulence genes was determined by growingcultures to an OD₆₀₀ of 0.8 in LB broth with 0.2 percent arabinose andthen adding 30 μg chloramphenicol/ml (37) for Crp, Fur and PhoP and 200μg chloramphenicol/ml for RpoS (38) to arrest further protein synthesis.As can be seen by the results presented in FIG. 28, the Crp, Fur andPhoP proteins are very stable and not significantly degraded, whereasthe RpoS protein displays no stability in the log phase (39). However,the RpoS protein seemed to be stable when 50 μg chloramphenicol/ml wasadded to saturated overnight stationery phase cultures. When thesemutant strains were grown in Nutrient broth with 0.2 percent arabinoseto an OD₆₀₀ of 0.8 and then diluted 1 to 4 into Nutrient broth with noadded arabinose and these 1 to 4 dilutions continued after each cultureagain reached an OD₆₀₀ of 0.8, we observed no significant reductions inthe amounts of Crp, Fur and PhoP proteins until a final dilution of 1 to16 with an arabinose concentration of 0.0125 percent or until a finaldilution of 1 to 64 with an arabinose concentration of 0.003125 percent.Thereafter the amount of these proteins decreased by a factor of fourfor each subsequent 1 to 4 dilution of the culture (FIG. 29). In thecase of RpoS protein, we observed a significant amount of reductionwithin a dilution of 1 to 4 with an arabinose concentration of 0.05percent (FIG. 29). The decline in the amounts of these proteins in vivowould be expected to be more accelerated since there is no arabinosepresent in tissues upon invasion of Salmonella into the GALT. In otherexperiments, strains grown in Nutrient broth with 0.2 percent arabinosewere sedimented by centrifugation and resuspended at a densityone-fourth of the original culture. In this case after growth to theoriginal density, the amounts of each of the four virulence geneproteins was three to four times less than in the culture grown witharabinose. In other experiments, it was determined that the levels ofFur, PhoP, RpoS and Crp synthesis were nearly the same when mutantcultures were grown in LB broth with either 0.05 percent or 0.2 percentarabinose.

Attenuation of Mutant Strains in Orally Immunized Female BALB/c Mice.

Levels of attenuation were evaluated in S. typhimurium UK-1 strains withdifferent araC P_(BAD) regulated virulence nucleic acid sequences byoral inoculation of female BALB/c mice with doses approximating 10⁷, 10⁸and 10⁹ CFU from cultures grown in LB broth with 0.0, 0.05 and 0.2percent arabinose. It should be noted, that LB broth contains arabinosein the yeast extract equivalent to a concentration of 0.003 percentbased on mass spec analysis. The collective results presented in Table 3indicate that the strains with the ΔP_(phoPQ107)::TT araC P_(BAD) phoPQ,ΔP_(rpoS183)::TT araC P_(BAD) rpoS and ΔP_(crp527)::TT araC P_(BAD) crpdeletion-insertion mutations were highly attenuated whereas the strainwith the ΔP_(fur33)::TT araC P_(BAD) fur mutation was less attenuated.In this regard, a higher level of attenuation was noted when χ8848 wasgrown in LB broth with no added arabinose and a greater virulence whengrown in LB broth with 0.2 percent arabinose. It is evident, however,from the collective results (Table 3) that attenuation develops as theproducts of the fur, phoPQ, rpoS and/or crp nucleic acid sequences arediluted at each cell division.

TABLE 3 Attenuation of mutant strains in orally immunized female BALB/cmice ^(a) Survivors/ Percent/ Strain Genotype Dose ^(b) range totalsurvivors χ8848 ΔP_(fur33) 9.0 × 10⁶-2.2 × 10⁹ 138/189 73.0 χ8918ΔP_(phoPQ107) 9.0 × 10⁶-1.2 × 10⁹ 182/185 98.4 χ8956 ΔP_(rpoS183) 9.4 ×10⁶-1.5 × 10⁹ 179/184 97.3 χ9021 ΔP_(crp527) 9.5 × 10⁶-1.5 × 10⁹ 163/16499.4 ^(a) Mice were seven to eight weeks of age. Bacterial strains weregrown in LB broth with 0, 0.05 or 0.2 percent arabinose that did nothave a significant effect on levels of attenuation on strains with theΔP_(phoPQ107)::TT araC P_(BAD) phoPQ, ΔP_(rpoS183)::TT araC P_(BAD) rpoSand ΔP_(crp527)::TT araC P_(BAD) crp deletion-insertion mutations butdid effect the results for χ8848 with the ΔP_(fur33)::TT araC P_(BAD)fur mutation (see Results). ^(b) Doses are in CFU.Abilities of Orally Administered Strains with araC P_(BAD) RegulatedVirulence Nucleic Acid Sequences to Induce Protective Immunity to OralChallenge with Wild-Type S. typhimurium UK-1.

Strains with each of the araC P_(BAD) regulated virulence nucleic acidsequences were next evaluated for induction of protective immunityagainst a challenge with the highly virulent S. typhimurium UK-1 strainχ3761 (oral LD₅₀ of 1-2×10⁴ CFU). The results in Table 4 reveal thatχ8848 with the ΔP_(fur33)::TT araC P_(BAD) fur mutation displayed somevirulence even at low doses when grown in LB broth with 0.2 percentarabinose. However, for immunizing doses of 10⁷ CFU and higher 100percent of the survivors developed protective immunity to challengeswith 10⁸ and 10⁹ CFU doses of χ3761. Thus the ΔP_(fur33)::TT araCP_(BAD) fur mutation while displaying moderate attenuation is highlyimmunogenic. This is a very important attribute of an attenuatingmutation to include in a vaccine strain. It was previously reported (40)that χ8848 with the ΔP_(fur33)::TT araC P_(BAD) fur mutation wascompletely attenuated even at high 10⁹ CFU doses when grown in LB brothwith no added arabinose. This observation implies that production of toomuch Fur protein may diminish attenuation.

TABLE 4 Oral immunization of mice with χ8848 (ΔP_(fur33)) and withsurvivors challenged orally with wild-type χ3761 thirty days later ^(a)Survivors/ Survivors/ Immunizing dose ^(b) total Challenge dose ^(b)total Exp. 1 Exp. 2 Exp. 1 Exp. 2 Exp. 1 Exp. 2 Exp. 1 Exp. 2 9.0 × 10⁸1.1 × 10⁹  6/10 4/10 1.0 × 10⁹ 1.5 × 10⁹ 3/3 2/2 1.0 × 10⁸ 1.5 × 10⁸ 3/32/2 9.0 × 10⁷ 1.1 × 10⁸  7/10 7/10 1.0 × 10⁹ 1.5 × 10⁹ 2/2 4/4 1.0 × 10⁸1.5 × 10⁸ 5/5 3/3 9.0 × 10⁶ 1.1 × 10⁷  7/10 5/10 1.0 × 10⁹ 1.5 × 10⁹ 4/42/2 1.0 × 10⁸ 1.5 × 10⁸ 3/3 3/3 9.0 × 10⁵ 1.1 × 10⁶  5/10 8/10 1.0 × 10⁹1.5 × 10⁹ 1/2 1/4 1.0 × 10⁸ 1.5 × 10⁸ 0/3 2/4 9.0 × 10⁴ 1.1 × 10⁵ 10/107/10 1.0 × 10⁹ 1.5 × 10⁹ 0/5 2/4 1.0 × 10⁸ 1.5 × 10⁸ 0/5 3/3 Total (alldoses) 66/100 45/66 68.2% Total (10⁷-10⁹ doses) 36/60  36/36  100% ^(a)Female BALB/c mice were six to eight weeks of age. χ8848 was grown in LBbroth with 0.2% arabinose. ^(b) Doses are in CFU.

The results in Table 5 reveal that χ8918 with the ΔP_(phoPQ107)::TT araCP_(BAD) phoPQ deletion-insertion mutation is very attenuated butdisplays more moderate immunogenicity in regard to inducing protectionagainst challenge with χ3761. These results suggest that some of theattenuation may be due to a reduced ability of χ8918 to effectivelycolonize lymphoid tissues, quite possibly due to the over expression ofthe phoPQ nucleic acid sequences when χ8918 is grown in LB broth with0.2 percent arabinose. In accord with this expectation, χ8918 is betterable to colonize Peyer's patches, mesenteric lymph nodes and spleens inorally immunized mice when grown in LB broth without added arabinosethan when grown in LB broth with 0.2 percent arabinose. Nevertheless,χ8918 is still less capable of colonizing these lymphoid tissues thanχ9021 with the ΔP_(crp527)::TT araC P_(BAD) crp deletion-insertionmutation, which colonizes equally well independent of arabinoseconcentration in the LB broth.

TABLE 5 Oral immunization of mice with χ8918 (ΔP_(phoPQ107)) and withsurvivors challenged orally with wild-type χ3761 thirty days later ^(a)Survivors/ Survivors/ Immunizing dose ^(b) total Challenge dose ^(b)total Exp. 1 Exp. 2 Exp. 1 Exp. 2 Exp. 1 Exp. 2 Exp. 1 Exp. 2 9.0 × 10⁸1.2 × 10⁹ 10/10  9/10 1.0 × 10⁹ 1.5 × 10⁹ 4/5 5/5 1.0 × 10⁸ 1.5 × 10⁸4/5 4/4 9.0 × 10⁷ 1.2 × 10⁸ 10/10 10/10 1.0 × 10⁹ 1.5 × 10⁹ 3/5 4/5 1.0× 10⁸ 1.5 × 10⁸ 3/5 5/5 9.0 × 10⁶ 1.2 × 10⁷ 10/10  9/10 1.0 × 10⁹ 1.5 ×10⁹ 2/4 1/4 1.0 × 10⁸ 1.5 × 10⁸ 2/5 2/5 9.0 × 10⁵ 1.1 × 10⁶ 10/10 10/101.0 × 10⁹ 1.5 × 10⁹ 3/5 0/5 1.0 × 10⁸ 1.5 × 10⁸ 0/5 3/5 9.0 × 10⁴ 1.1 ×10⁵ 10/10 10/10 1.0 × 10⁹ 1.5 × 10⁹ 0/5 0/5 1.0 × 10⁸ 1.5 × 10⁸ 0/5 0/5Total (all doses)  98/100 45/98 45.9% Total (10⁷-10⁹ doses) 58/60 39/5867.2% ^(a) Female BALB/c mice were six to eight weeks of age. χ8918 wasgrown in LB broth with 0.2% arabinose. ^(b) Doses are CFU.

The results in Table 6 confirm the oral avirulence of χ8956 with theΔP_(rpoS183)::TT araC P_(BAD) rpoS deletion-insertion mutation. However,the two experiments give very different results on the ability of thisstrain to induce protective immunity to oral challenge with wild-type S.typhimurium. We therefore repeated the experiment giving oral doses ofχ8956 (ΔP_(rpoS183)) of 1.4×(10⁷, 10⁸ and 10⁹) CFU with 15 survivors ateach dose and after challenge with 3.1×10⁹ CFU of χ3761 observed 13, 13and 14 survivors, respectively, out of 15 mice challenged. It thusappears that the data in the second experiment in Table 6 are moreindicative of the correct attenuating and immunogenic phenotype. Nodifferences in results were observed when χ8956 (ΔP_(rpoS183)) was grownin LB broth with or without arabinose.

TABLE 6 Oral immunization of mice with χ8956 (ΔP_(rpoS183)) and withsurvivors challenged orally with wild-type-χ3761 thirty days later ^(a)Survivors/ Survivors/ Immunizing dose ^(b) total Challenge dose ^(b)total Exp. 1 Exp. 2 Exp. 1 Exp. 2 Exp. 1 Exp. 2 Exp. 1 Exp. 2 9.4 × 10⁸1.5 × 10⁹  9/10  9/10 1.0 × 10⁹ 1.5 × 10⁹ 0/4 4/4 1.0 × 10⁸ 1.5 × 10⁸1/5 5/5 9.4 × 10⁷ 1.5 × 10⁸ 10/10 10/10 1.0 × 10⁹ 1.5 × 10⁹ 0/5 5/5 1.0× 10⁸ 1.5 × 10⁸ 0/5 3/5 9.4 × 10⁶ 1.5 × 10⁷ 10/10  9/10 1.0 × 10⁹ 1.5 ×10⁹ 2/5 5/5 1.0 × 10⁸ 1.5 × 10⁸ 0/5 2/4 9.4 × 10⁵ 1.5 × 10⁶ 10/10 10/101.0 × 10⁹ 1.5 × 10⁹ 0/5 4/5 1.0 × 10⁸ 1.5 × 10⁸ 0/5 2/5 9.4 × 10⁴ 1.5 ×10⁵ 10/10 10/10 1.0 × 10⁹ 1.5 × 10⁹ 0/5 0/5 1.0 × 10⁸ 1.5 × 10⁸ 0/5 0/5Total (all doses)  97/100 33/97 34.0% Total (10⁷-10⁹ doses) 57/60 27/5747.4% ^(a) Female BALB/c mice were six to eight weeks of age. χ8956 wasgrown in LB broth with 0.2% arabinose. ^(b) Doses are in CFU.

The results in Table 7 indicate that χ9021 with the ΔP_(crp527)::TT araCP_(BAD) crp deletion-insertion mutation is both highly attenuated andalso very immunogenic. Neither of these attributes was altered when thestrain was grown in LB broth with or without arabinose.

TABLE 7 Oral immunization of mice with χ9021 (ΔP_(crp527)) and withsurvivors challenged orally with wild-type χ3761 thirty days later ^(a)Survivors/ Survivors/ Immunizing dose ^(b) total Challenge dose ^(b)total Exp. 1 Exp. 2 Exp. 1 Exp. 2 Exp. 1 Exp. 2 Exp. 1 Exp. 2 9.5 × 10⁸1.6 × 10⁹ 10/10 10/10 1.0 × 10⁹ 1.5 × 10⁹ 5/5 5/5 1.0 × 10⁸ 1.5 × 10⁸5/5 5/5 9.5 × 10⁷ 1.6 × 10⁸ 10/10 10/10 1.0 × 10⁹ 1.5 × 10⁹ 5/5 5/5 1.0× 10⁸ 1.5 × 10⁸ 5/5 5/5 9.5 × 10⁶ 1.6 × 10⁷ 10/10 10/10 1.0 × 10⁹ 1.5 ×10⁹ 4/5 5/5 1.0 × 10⁸ 1.5 × 10⁸ 5/5 5/5 9.5 × 10⁵ 1.6 × 10⁶ 10/10  9/101.0 × 10⁹ 1.5 × 10⁹ 5/5 3/5 1.0 × 10⁸ 1.5 × 10⁸ 3/5 2/4 9.5 × 10⁴ 1.6 ×10⁵ 10/10 10/10 1.0 × 10⁹ — 4/5 — 1.0 × 10⁸ — 3/5 — Total (all doses) 99/100 78/89 87.6% Total (10⁷-10⁹ doses) 60/60 59/60 98.3% ^(a) FemaleBALB/c mice were six to eight weeks of age. χ9021 was grown in LB brothwith 0.2% arabinose. ^(b) Doses are in CFU.Alterations in Strains with the ΔP_(fur)::TT araC P_(BAD) Fur andΔP_(phoPQ)::TT araC P_(BAD) phoPQ Deletion-Insertion Mutations toIncrease the Attenuation of the Former and Increase the Immunogenicityof the Latter.

As noted above, χ8848 with the ΔP_(fur33)::TT araC P_(BAD) fur mutationwas more attenuated when grown in LB broth without arabinose and morevirulent when grown in LB broth with 0.2 percent arabinose prior to oralinoculation of mice. This implied that overproduction of Fur, whichwould require more cell divisions in vivo to dilute out, reducedattenuation without adversely altering immunogenicity in mice survivingimmunization. Consequently, two derivatives were constructed in whichthe ATG start codon for the fur nucleic acid sequence was changed toGTG, and in one of these, the SD sequence was changed from AGGA to AAGG.The structure of these two mutations, ΔP_(fur77)::TT araC P_(BAD) furand ΔP_(fur81)::TT araC P_(BAD) fur, are diagrammed in FIG. 8. χ9273with the ΔP_(fur77)::TT araC P_(BAD) fur mutation and χ9269 with theΔP_(fur81)::TT araC P_(BAD) fur mutation both synthesize much less Furas reveled by western blot analysis when grown in LB broth with 0.2percent arabinose than does χ8848 with the ΔP_(fur33)::TT araC P_(BAD)fur mutation.

It was also noted above that the immunogenicity of χ8918 with theΔP_(phoPQ107)::TT araC P_(BAD) phoPQ mutation was decreased when thestrain was grown in LB broth with 0.2 percent arabinose although itsattenuation was independent of the arabinose concentration in LB broth.This implied that over production of PhoP and/or PhoQ decreasedinduction of immunity to challenge. This inference was also supported bystudies that demonstrated that χ8918 was less able to colonize Peyer'spatches, mesenteric lymph nodes and spleen when grown in LB broth with0.2 percent arabinose than when grown with no added arabinose. Twoderivatives were therefore constructed in which the ATG start codon forthe phoP nucleic acid sequence was changed to GTG and in one of thesealso changed the SD sequence from AGGA to AAGG. The structure of thesetwo mutations, ΔP_(phoPQ173)::TT araC P_(BAD) phoPQ andΔP_(phoPQ177)::TT araC P_(BAD) phoPQ, are diagrammed in FIG. 9. χ9382with the ΔP_(phoPQ173)::TT araC P_(BAD) phoPQ mutation and χ9383 withthe ΔP_(phoPQ177)::TT araC P_(BAD) phoPQ mutation both synthesize muchless PhoP as reveled by western blot analysis when grown in LB brothwith 0.2 percent arabinose than does χ8918 with the ΔP_(phoPQ107)::TTaraC P_(BAD) phoPQ mutation.

Table 8 below contains results that demonstrate the high immunogenicityof χ9273 with the ΔP_(fur77)::TT araC P_(BAD) fur mutation and χ9269with the ΔP_(fur81)::TT araC P_(BAD) fur mutation with χ9269 with theΔP_(fur81)::TT araC P_(BAD) fur mutation demonstrating much betterattenuation when grown in LB broth with 0.2 percent arabinose. The datain Table 8 also indicates that both χ9382 with the ΔP_(phoPQ173)::TTaraC P_(BAD) phoPQ mutation and χ9383 with the ΔP_(phoPQ177)::TT araCP_(BAD) phoPQ mutation are completely attenuated when grown in LB brothwith 0.2 percent arabinose and display essentially the sameimmunogenicity that is much improved over that exhibited by χ8918 withthe ΔP_(phoPQ107)::TT araC P_(BAD) phoPQ mutation when it is grown in LBbroth with 0.2 percent arabinose.

TABLE 8 Oral immunization of mice with strains with modified ΔP_(fur)and ΔP_(phoPQ) mutations and with survivors challenged orally withwild-type χ3761 thirty days later.^(a) Immunizing Survivors/ ChallengeSurvivors/ Strain^(b) Genotype dose^(c) total dose^(c) total χ9273ΔP_(fur77) 1.5 × 10⁹  6/10 1.7 × 10⁹ 6/6 1.7 × 10⁹  6/10 1.6 × 10⁹ 6/6χ9269 ΔP_(fur81) 1.0 × 10⁹ 11/15 8.7 × 10⁸ 11/11 1.0 × 10⁸ 15/15 8.7 ×10⁸ 15/15 1.8 × 10⁹ 10/10 1.3 × 10⁹ 10/10 1.7 × 10⁹ 19/20 1.6 × 10⁹19/19 χ9382 ΔP_(phoPQ173) 1.0 × 10⁹ 15/15 1.8 × 10⁹ 11/15 1.0 × 10⁸15/15 1.8 × 10⁹ 11/15 1.0 × 10⁷ 15/15 1.8 × 10⁹ 12/15 χ9383ΔP_(phoPQ177) 1.1 × 10⁹ 15/15 1.8 × 10⁹ 10/15 1.1 × 10⁸ 15/15 1.8 × 10⁹11/15 1.1 × 10⁷ 15/15 1.8 × 10⁹ 13/15 ^(a)Female BALB/c mice were six toeight weeks of age. Strains were grown in LB broth with no addedarabinose or with 0.05% or 0.2% arabinose with no significantdifferences noted. ^(b)χ9382 and χ9383 have the ΔaraBAD23 deletion(Table 1) in addition to the ΔP_(phoPQ) insertion-deletion mutations theΔaraBAD23 deletion (Table 5). ^(c)Doses are in CFU.Abilities of Intraperitoneally Administered Strains with araC P_(BAD)Regulated Virulence Nucleic Acid Sequences to Induce Protective Immunityto Oral Challenge with Wild-Type S. typhimurium UK-1.

Although the vaccines were designed for oral administration, it wasworthwhile to determine if strains with these mutations, whenadministered intraperitoneally (i.p.), would also display attenuationand induce immunity to challenge with orally administered wild-typeχ3761. The S. typhimurium UK-1 strain χ3761 has an LD₅₀ by the i.p.route of less than 10 CFU. Table 9 demonstrates that strains withΔP_(fur)::TT araC P_(BAD) fur mutations retain considerable virulence bythis route of administration although χ9269 with the ΔP_(fur81)::TT araCP_(BAD) fur mutation displays the highest attenuation of the threestrains evaluated and yet induces complete protective immunity to allsurvivors when challenged with about 10⁹ CFU of χ3761. χ8918 with theΔP_(phoPQ107)::TT araC P_(BAD) phoPQ mutation displays fairly goodattenuation by this route. On the other hand, χ8956 with theΔP_(rpoS183)::TT araC P_(BAD) rpoS mutation and χ9021 with theΔP_(crp527)::TT araC P_(BAD) crp mutation are the most attenuated andinduce a very high level of protective immunity when delivered at i.pdoses in the 10² to 10⁴ CFU range (Table 9).

TABLE 9 Intraperitoneal immunization of mice with strains with variousdeletion-insertion mutations conferring regulated delayed oralattenuation and with survivors orally challenged with wild-type χ3761thirty days later. Immunizing Survivors/ Challenge Survivors/ StrainGenotype dose^(b) total dose^(b) total χ8848 ΔP_(fur33) 1.2 × 10⁴ 0/5 —1.2 × 10³ 0/5 — 1.2 × 10² 0/5 — 1.2 × 10¹ 0/5 — χ9273 ΔP_(fur77) 1.6 ×10⁴ 0/5 1.6 × 10³ 0/5 1.6 × 10² 0/5 1.6 × 10¹ 1/5 1.6 × 10⁹ 1/1 χ9269ΔP_(fur81) 1.7 × 10⁴  1/10 1.6 × 10⁹ 1/1 1.7 × 10³  7/10 1.6 × 10⁹ 7/71.7 × 10²  4/10 1.6 × 10⁹ 4/4 1.7 × 10¹  6/10 1.6 × 10⁹ 6/6 χ8918ΔP_(phoPQ107) 1.2 × 10⁵ 0/5 — 9.6 × 10⁴  6/10 1.5 × 10⁹ 4/6 1.2 × 10⁴5/5 9.6 × 10⁸ 5/5 9.6 × 10³  8/10 1.5 × 10⁹ 6/8 1.3 × 10³ 5/5 9.6 × 10⁸2/5 9.6 × 10² 5/5 1.5 × 10⁹ 2/5 1.2 × 10² 5/5 9.6 × 10⁸ 3/5 χ8956ΔP_(rpoS183) 1.5 × 10⁴ 5/5 1.0 × 10⁹ 5/5 1.5 × 10³ 5/5 1.0 × 10⁹ 5/5 1.5× 10² 5/5 1.0 × 10⁹ 3/5 1.5 × 10¹ 5/5 1.0 × 10⁹ 3/5 χ9021 ΔP_(crp527)1.4 × 10⁵ 0/5 — 1.4 × 10⁴  4/10 1.5 × 10⁹ 4/4 1.4 × 10³ 10/10 1.5 × 10⁹10/10 1.4 × 10² 10/10 1.5 × 10⁹ 10/10 1.4 × 10¹  9/10 1.5 × 10⁹ 8/9^(a)Female BALB/C mice were six to eight weeks of age. All strains weregrown in LB broth with 0.2% arabinose. ^(b)Doses are in CFU.Enhanced Control Over araC P_(BAD) Regulated Virulence Nucleic AcidSequences In Vivo by Inclusion of the ΔP_(crp527)::TT araC P_(BAD) CrpMutation.

Maximum levels of transcription of nucleic acid sequences regulated bythe araC P_(BAD) system require not only arabinose to interact with theAraC protein but also the Crp protein (41). Thus the ΔP_(crp527)::TTaraC P_(BAD) crp mutation was included in vaccine strains whenever otheraraC P_(BAD) regulated nucleic acid sequences are included. The benefitof this addition is readily observed by the results previously presentedin FIG. 7 that demonstrate this tighter regulation in the absence ofarabinose in strains that also have the ΔP_(crp527)::TT araC P_(BAD) crpmutation. This also acts as a backup and should enhance safety andefficacy of vaccine strains.

Means for delay in the in vivo timing of onset of regulated delayedattenuation. As noted by Guzman et al. (32), the inclusion of mutationsthat abolish utilization of arabinose can prolong expression of nucleicacid sequences under the control of the araC P_(BAD) system. Onset ofattenuation can therefore by delayed by including ΔaraBAD23, whichprevents use of arabinose retained in the cell cytoplasm at the time oforal immunization, and/or ΔaraE25 that enhances retention of arabinose.These mutations are diagrammed in FIG. 27.

Discussion for Example 2

Four different means have been described to achieve regulated delayedattenuation of S. typhimurium vaccine strains such that vaccines at thetime of immunization will be better able to withstand the host defenseimposed stresses following oral immunization. Some of these constructshave been modified to optimize attenuation and improve immunogenicity.Although comparative studies with vaccine strains having defineddeletion mutations in the fur, phoPQ, rpoS and crp nucleic acidsequences might resolve doubt, such comparative studies become difficultto justify based on animal use. These mutations are being included instrains with multiple attenuating mutations.

REFERENCES FOR EXAMPLE 2

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Bang I S, Audia J P, Park Y K, & Foster J W (2002) Autoinduction    of the ompR response regulator by acid shock and control of the    Salmonella enterica acid tolerance response. Mol Microbiol 44:    1235-1250.-   11. Bang I S, Kim B H, Foster J W, & Park Y K (2000) OmpR regulates    the stationary-phase acid tolerance response of Salmonella enterica    serovar Typhimurium. J Bacteriol 182: 2245-2252.-   12. Prouty A M & Gunn J S (2000) Salmonella enterica serovar    Typhimurium invasion is repressed in the presence of bile. Infect    Immun 68: 6763-6769.-   13. van Velkinburgh J C & Gunn J S (1999) PhoP-PhoQ-regulated loci    are required for enhanced bile resistance in Salmonella spp. Infect    Immun 67: 1614-1622.-   14. Gunn J S (2000) Mechanisms of bacterial resistance and response    to bile. Microbes Infect 2: 907-913.-   15. Curtiss R, III & Hassan J O (1996) Nonrecombinant and    recombinant avirulent Salmonella vaccines for poultry. 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Kang H Y, Dozois C M, Tinge S A, Lee T H, & Curtiss, R.,    III (2002) Transduction-mediated transfer of unmarked deletion and    point mutations through use of counterselectable suicide vectors. J    Bacteriol 184: 307-312.-   27. Daigle F, Graham J E, & Curtiss, R., III (2001) Identification    of Salmonella typhi genes expressed within macrophages by selective    capture of transcribed sequences (SCOTS). Mol Microbiol 41:    1211-1222.-   28. Galan J E & Curtiss R, III (1989) Cloning and molecular    characterization of genes whose products allow Salmonella    typhimurium to penetrate tissue culture cells. Proc Natl Acad Sci    USA 86: 6383-6387.-   29. Curtiss R, III & Kelly S M (1987) Salmonella typhimurium    deletion mutants lacking adenylate cyclase and cyclic AMP receptor    protein are avirulent and immunogenic. Infect Immun 55: 3035-3043.-   30. Gulig P A & Curtiss R, III (1987) Plasmid-associated virulence    of Salmonella typhimurium. Infect Immun 55: 2891-2901.-   31. 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Example 3: Improved Immune Responses Induced by RASVs with RegulatedDelayed Attenuation and Regulated Delayed Synthesis of ProtectiveAntigen

Generating a Salmonella strain that is safe and also retains itsimmunogenicity is the biggest challenge in the development of livevaccine candidates (1). An ideal Salmonella vaccine strain shouldexhibit wild-type abilities to withstand all stresses (enzymatic, acid,osmotic, ionic, etc.) and host defenses (bile, antibacterial peptides,etc.) encountered following oral or intranasal immunization and shouldexhibit wild-type abilities to colonize and invade host lymphoid tissueswhile remaining avirulent. A variety of attenuated Salmonella strainshave been used as live vaccines to induce mucosal and systemic immunityagainst either the carrier itself or to a vectored antigen (2). Morerecently developed Salmonella vaccine strains carry defined nonrevertingmutations that fall into two general categories: metabolic functions andvirulence factors (3). Different means for attenuating Salmonella havebeen investigated to develop ideal immune responses (4, 5). Manypreviously utilized means for Salmonella attenuation either reducedvaccine survival due to host-induced stresses and/or reducedcolonization of lymphoid effector tissues leading to less than optimalimmunogenicity (6, 7). To circumvent these problems, a system forregulated delayed in vivo expression of attenuation has been developed(8). Thus vaccine strains are phenotypically wild-type for host invasionat the time of immunization and become attenuated after colonization ofhost tissues (8).

PspA is an important virulence factor found on the surface of allpneumococci (9). It plays a role in colonization of the host andcontributes to the ability of pneumococcus to cause invasive disease(10). The N-terminal half of the protein is the α-helical domain whichcontains protective epitopes based on immunization studies with the fulllength and truncated PspA fragments (11, 12). Humans naturally infectedor colonized with pneumococcus, develop anti-PspA antibodies in bothserum and mucosal secretions, with antibody to the α-helical domain ofPspA also implicated in preventing pneumococcal carriage.

Previous work has demonstrated that oral vaccination of mice with a ΔcrpSalmonella vaccine strain expressing a secreted PspA fusion proteincould protect the immunized mice from virulent S. pneumoniae WU2challenge (13). The protection rate was about 60% against a 50 LD₅₀challenge (13). To increase the effective protective immunity against S.pneumoniae, we designed and constructed a new generation of Salmonellaenterica serovar Typhimurium strains with delayed regulated attenuation(see Example 2) and for one strain with regulated delayed expression ofantigen encoding sequences in vivo (Example 1).

In this example, the immunogenicity is evaluated of two new attenuatedS. typhimurium strains transformed with Asd⁺ balanced-lethal plasmidsencoding a secreted form of the α-helical region of PspA. Antibodyresponses, cytokine responses and protective immunity against S.pneumoniae WU2 challenge were evaluated. The results attained confirmthe hypothesis that vaccine strains with regulated delayed in vivoattenuation including the strain that also exhibited regulated delayedprotective antigen synthesis confer a more superior immune response thana vaccine strain with a more traditional means of attenuation.

Materials and Methods for Example 3

Bacterial Strains, Plasmids, Media, and Growth Conditions:

The bacterial strains and plasmids used in this example are listed inTable 1 and 2, respectively. Bacteriophage P22HT int was used forgeneralized transduction. S. typhimurium cultures were grown at 37° C.in LB broth or on LB agar (14). For animal experiments,plasmid-containing χ9088 and χ9558 cultures were supplemented with 0.2%mannose or 0.2% mannose and 0.05% arabinose, respectively. No additionswere made to the media for growing plasmid-containing χ8133 cultures.MacConkey agar (Difco, Detroit, Mich.) supplemented with 1% sugar wasused for fermentation assays. DAP was added (50 μg/ml) for the growth ofAsd− strains (15). S. pneumoniae WU2 was cultured on brain heartinfusion agar containing 5% sheep blood or in Todd-Hewitt broth plus0.5% yeast extract (12).

Strain Construction and Characterization:

MacConkey agar supplemented with 1% maltose was used to confirm thephenotype of crp mutants (13). Chrome Azurol S (CAS) plates were used toconfirm the constitutive synthesis of siderophores characteristic of furmutants (16). The presence of the ΔasdA33 and ΔasdA16 mutations inSalmonella was confirmed by inability of the strain to grow on mediawithout DAP (15). Lipopolysaccharide (LPS) profiles of Salmonellastrains were examined as described (17). Plasmid stability wasdetermined as previously described (18). All plasmids were found to bestable for 50 generations of growth in the presence of DAP.

SDS-PAGE and Immunoblot Analyses:

Protein samples were boiled for 5 min and subsequently separated bySDS-PAGE. For immunoblotting, proteins separated by SDS-PAGE weretransferred to nitrocellulose membranes. After blocking membranes with3% skim milk in 10 mM Tris-0.9% NaCl (pH 7.4), PspA was detected withrabbit polycolonal antibody specific for PspA (University of Alabama atBirmingham) followed by the addition of an AP-conjugated goatanti-rabbit immunoglobulin G (IgG) (Sigma). Immunoreactive bands werevisualized by the addition of BCIP/NBT solution (Sigma). The reactionwas stopped after 2 min by washing with large volumes of deionized waterseveral times.

Immunization of Mice:

Female BALB/c mice, 6-7 weeks old, were obtained from Charles RiverLaboratories. All animal procedures were approved by the Arizona StateUniversity Animal Care and Use Committee. Mice were acclimated for 7days before starting experiments.

Recombinant attenuated Salmonella vaccine (RASV) strains were grownstatically overnight in LB broth containing the appropriate supplementsat 37° C. The following day, an overnight culture of 1 ml was inoculatedinto 100 ml of LB broth containing the appropriate supplements and grownwith aeration at 37° C. to an OD₆₀₀ of 0.8 to 0.9. Cells were pelletedby centrifugation at room temperature (6,000×g for 15 min), and thepellet resuspended in 1 ml of buffered saline with gelatin (BSG). Todetermine the titer of RASV strains used to inoculate mice, dilutions ofthe RASV strains were plated onto MacConkey agar supplemented with 1%lactose. Mice were orally inoculated with 20 μl of BSG containing 1×10⁹CFU of an RASV strain. Blood was obtained by mandibular vein puncture atbiweekly intervals. Following centrifugation, the serum was removed fromthe whole blood and stored at −20° C.

Antigen Preparation:

rPspA protein and S. typhimurium outer membrane proteins (SOMPs) werepurified as described (13). S. typhimurium LPS was obtained from Sigma.The rPspA clone and purified protein were kind gifts from Dr. SusanHollingshead at the University of Alabama at Birmingham (19).

Enzyme Linked Immunosorbent Assay (ELISA):

ELISA was used to assay antibodies in serum to S. typhimurium LPS, SOMPsand to rPspA. Polystyrene 96-well flat-bottom microtiter plates(Dynatech Laboratories Inc., Chantilly, Va.) were coated with LPS (100ng/well; Sigma), SOMP (100 ng/well, our lab), or purified rPspA (100ng/well). Antigens suspended in sodium carbonate-bicarbonate coatingbuffer (pH 9.6) were applied with 100-μl volumes in each well. Plateswere incubated overnight at 4° C. Free binding sites were blocked withphosphate-buffered saline (pH 7.4) containing 0.1% Tween 20, and 1%bovine serum albumin. A 100-μl volume of series diluted sample was addedto individual wells in triplicate and incubated for 1 h at 37° C. Plateswere treated with biotinylated goat anti-mouse IgG, IgG1, or IgG2a(Southern Biotechnology Inc., Birmingham, Ala.) Wells were developedwith streptavidin-alkaline phosphatase conjugate (SouthernBiotechnology) followed by p-nitrophenylphosphate substrate (Sigma) indiethanolamine buffer (pH 9.8). Color development (absorbance) wasrecorded at 405 nm using an automated ELISA plate (model EL311SX;Biotek, Winooski, Vt.). Absorbance readings 0.1 higher than PBS controlvalues were considered positive reactions.

Passive Transfer of Cells and Sera:

At week 12, sera and spleen cells were harvested from 5 mice per group.The sera were pooled and CD4⁺ T cells were isolated using T-cellenrichment columns (R&D Systems Inc, Minneapolis, Minn.), according tothe manufacturer's instructions. Spleen cells (1×10⁷) or purified CD4⁺ Tcells (5×10⁶) were suspended in PBS and injected into the lateral tailveins of naive, syngeneic BALB/c mice. Naïve syngeneic BALB/c micereceived 100 μl of serum from a different group of mice through the tailvein. All groups were challenged intraperitoneally after 12 h with 5×10⁴CFU of S. pneumoniae in 200 μl of BSG.

IL-4 and IFN-γ ELISPOTs:

At week 8, spleen cells were harvested from 3 mice of each group.ELISPOTs were performed as previously described (20). Briefly, PVDFmembrane plates (Millipore, Bedford, Mass., USA) were pre-wetted withethyl alcohol, washed with sterile H₂O and coated with 100 μl of mAbsIL-4 or IFN-γ (BD PharMingen, San Diego, Calif.) at 2 μg/ml, in PBSovernight at 4° C. The wells were washed with PBS and blocked with RPMIwith 10% FCS. After that, 50 μl cell medium (RPMI-1640 supplemented with10% FCS, 2 mM L-glutamine, 100 IU/ml penicillin and streptomycin and 1%HEPES, with or without stimuli and 50 μl of cells (100,000 per well) incell medium were added per well and incubated in the plates overnight in5% CO₂ at 37° C. The next day, the cell suspensions were discarded andthe plates washed with PBS. Biotinylated mAb IL-4 or IFN-γ (BDPharMingen, San Diego, Calif.) at 0.5 μg/ml in PBS with 1% FCS was addedand incubated at room temperature for 2 h. After washing with PBS, 100μl/well of avidin peroxidase diluted 1:1000 (v/v) in PBS-T containing 1%FCS were added followed by incubation for 1 hour at room temperature.AEC (3-amina-9-ethycarbazole) substrate was prepared according tomanufacturer's (Vector Laboratories, Burlingame, Calif.) specifications,and 100 μl of substrate was added per well. Spots were developed for 15minutes at room temperature. Plates were dried and analyzed by using anautomated CTL ELISPOT Reader System (Cellular Technology LTD, Cleveland,Ohio).

Measurement of Cytokine Concentrations:

Cytokine concentrations were determined using the Bio-Plex Protein ArraySystem (Bio-Rad, Hercules, Calif., USA). Cytokine-specificantibody-coated beads (Bio-Rad) were used for these experiments. Theassay quantitates cytokines over a broad range (2-32,000 μg/ml) andeliminates multiple dilutions of high-concentration samples. The sampleswere prepared and incubated with the antibody-coupled beads for 1 h withcontinuous shaking. The beads were washed three times with wash bufferto remove unbound protein and then incubated with biotinylated detectioncytokine-specific antibody for 1 h with continuous shaking. The beadswere washed once more and were then incubated withstreptavidin-phycoerythrin for 10 min. After incubation, the beads werewashed and resuspended in assay buffer, and the constituents of eachwell were drawn up into the flow-based Bio-Plex Suspension Array System,which identifies each different color bead as a population of proteinand quantifies each protein target based on secondary antibodyfluorescence. Cytokine concentrations were automatically calculated byBio-Plex Manager software using a standard curve derived from arecombinant cytokine standard. Many readings were made on each bead set,further validating the results.

Pneumococcal Challenge:

At week 12 the ability of the Salmonella-PspA vaccine to protectimmunized mice against S. pneumoniae was assessed by intraperitonealchallenge with 5×10⁴ CFU of S. pneumoniae WU2 in 200 μl of BSG (21). The50% lethal dose (LD₅₀) of S. pneumoniae WU2 in BALB/c mice was 2×10² CFUby intraperitoneal administration. Twenty-four hours afterintraperitoneal challenge, mice were marked and bled by mandibular veinpuncture, and blood samples with 10-fold serial dilutions in saline wereplated on brain heart infusion agar containing 5% sheep blood. Bacterialcolonies were enumerated after overnight incubation at 37° C.

Histological Examinations:

After challenge, mice were euthanized just before dying and survivorswere euthanized after 15 days of observation. Fixed lung, spleen andliver specimens were embedded in paraffin wax, sectioned, and stainedwith hematoxylin and eosin (H&E). Micrographs were taken with a digitalcamera.

Statistical Analysis:

Most data were expressed as means±standard error. The means wereevaluated with One-way ANOVA and LSD tests for multiple comparisonsamong groups. p<0.05 was considered statistically significant.

Regulated Attenuation of Fur, Crp and Pmi in χ9088 (ΔP_(fur33)::TT araCP_(BAD) Fur ΔPmi-2426 Δ(gmd-fcl)-26 ΔasdA33) and χ9558 (Δpmi-2426Δ(gmd-fcl)-26 ΔP_(fur81)::TT araC P_(BAD)fur ΔP_(crp527)::TT araCP_(BAD)Crp ΔasdA27::TT araC P_(BAD)c2 ΔaraE25 ΔaraBAD23 ΔrelA198::araCP_(BAD)lacl TT ΔsopB1925 ΔagfBAC811).

Regulated delayed attenuation attributes distinguish χ9088 and χ9558from other attenuated Salmonella strains due to a unique combination ofarabinose-regulated expression of Fur, Crp and mannose-regulatedexpression of O-antigen synthesis. Salmonella with pmi mutations areattenuated and immunogenic (22). Strains with the Δpmi-2426 mutationlack phosphomannose isomerase needed to interconvert fructose-6-P andmannose-6-P but synthesize a complete LPS O-antigen when grown in thepresence of mannose (FIG. 30). Note that LPS synthesis is dependent onthe addition of mannose when cells are grown in nutrient broth, butthere is enough mannose in LB broth to enable O-antigen production.

The other means used to achieve regulated delayed in vivo attenuationwas to replace the promoter/operator regions of the fur and crp nucleicacid sequences with the tightly-regulated, arabinose-dependent araCP_(BAD) activator-promoter. (FIG. 31) Growth of these mutant strains inthe presence of arabinose leads to transcription of the fur and crpnucleic acid sequences but nucleic acid sequence expression ceases inthe absence of arabinose. Since free arabinose is not found in mammaliantissues, the arabinose-regulated fur and crp nucleic acid sequences willnot be expressed.

Expression of rPspA in Salmonella.

The recombinant plasmid pYA3634 (pBR on) was constructed for theperiplasmic secretion of the α-helical region of the PspARx1 (8)(Table2). Plasmid pYA3493 (vector control) and pYA3634 (encoding P3-lactamase(bla)SS-pspA) were electroporated into S. typhimurium strains χ8133,χ9088 and χ9558. The RASV electroporants containing pYA3634 expressed aprotein with an approximate molecular mass of 37 kDa, the expected sizeof the Bla SS-PspA fusion protein encoded by pYA3634, that reactsspecifically with an anti-PspA polyclonal antibody (FIG. 32). Plasmidstability was evaluated as described in the Material and Methods.Plasmids were maintained and protein expression was shown to be stablewhen strains were grown in the presence of DAP for 50 nucleic acidsequencerations.

Antibody Responses in Mice after Oral Immunization with the RecombinantS. enterica Serovar Typhimurium Vaccines.

A dose of RASV χ8133(pYA3634) (1.9×10⁹ CFU), χ9088(pYA3634) (1.7×10⁹CFU), χ9558(pYA3634) (1.5×10⁹ CFU), χ8133(pYA3493) (1.8×10⁹ CFU),χ9088(pYA3493) (2.0×10⁹ CFU) or χ9558(pYA3493) (1.5×10⁹ CFU/mouse) wasorally administered to 7-week-old female BALB/c mice. Mice were boostedwith the same dose of the same strain eight weeks later. All immunizedmice survived, and no signs of disease were observed in the immunizedmice during the entire experimental period. The antibody responses toSalmonella LPS, SOMPs and rPspA in the sera of immunized mice weremeasured (FIG. 33). High serum IgG titers against all antigens,including PspA, were observed by 2 weeks after the primary immunization.The maximum preboost anti-LPS, -SOMP, and -rPspA IgG levels weredetected by 6 weeks post immunization. The serum anti-rPspA IgG antibodylevels of χ9558(pYA3634) and χ9088(pYA3634) immunized mice weresignificantly higher than the levels in χ8133(pYA3634) immunized mice(p<0.01, p<0.05).

IgG Isotype Analyses

The types of immune responses to rPspA were further examined bymeasuring the levels of IgG isotype subclasses IgG1 and IgG2a (FIG. 34).The Th1-helper cells direct cell-mediated immunity and promote classswitching to IgG2a, and Th2 cells provide potent help for B-cellantibody production and promote class switching to IgG1 (23, 24).Th1-type dominant immune responses are frequently observed afterimmunization with attenuated Salmonella (25-27). A Th1- and Th2-typemixed response was observed for the rPspA antigen. Although the IgG1levels were almost the same as IgG2a levels in the early phase, thelevel of anti-rPspA IgG2a isotype antibodies gradually increased afterboosting at 8 weeks. After 12 weeks postimmunization, the ratios ofIgG1: IgG2a are 1:8.3 for χ8133(pYA3634) immunized mice, 1:9.4 forχ9088(pYA3634) immunized mice and 1:1.5 for χ9558(pYA3634) immunizedmice. The serum anti-rPspA IgG1 and IgG2a antibody levels ofχ9558(pYA3634) and χ9088(pYA3634) immunized mice are both significantlyhigher than those of the χ8133(pYA3634) immunized mice (p<0.01) (FIG.34).

Antigen-Specific Stimulation of IL-4 or IFN-g Production.

ELISPOT was used to compare PspA antigen stimulation of IL-4 or IFN-gproduction by cells from spleens of immunized control mice (FIG. 35).Spleen lymphocytes from χ9088(pYA3634) immunized mice had (114.0±28.8)antigen specific IL-4 secreting cells per 10⁶ cells and (315.9±31.5)antigen specific IFN-g secreting cells per 10⁶ cells which were bothsignificantly higher than those of the spleen lymphocytes fromχ8133(pYA3634) immunized mice (p<0.001) The spleen lymphocytes fromχ9558(pYA3634) immunized mice had (108.1±90.25) antigen specific IL-4secreting cells per 10⁶ cells and (280.1±47.10) antigen specific IFN-gsecreting cells per 10⁶ cells which were also significantly higher thanthose of the spleen lymphocytes from χ8133(pYA3634) immunized mice(p<0.001).

Status of Systemic Cytokine Environment.

At 2 weeks after immunization, sera from each group of mice weresubjected to Bio-Plex analyses. The secretion profiles were compared(Table 10). The sera from χ8133(pYA3634), χ9088(pYA3634), χ9558(pYA3634)immunized mice have increased levels of cytokine concentrations comparedwith the BSG control group (p<0.01). χ9558(pYA3634) immunized mice havean increased level of cytokines including both Th1 and Th2 cytokinescompared with the χ8133(pYA3634) group (p<0.05), which suggested thatthe RASV strains caused mixed Th1 and Th2 responses and our regulateddelayed attenuation strain χ9558(pYA3634) stimulated stronger cellularimmunity and cytokine secretion than χ8133(pYA3634), which willfacilitate antigen presentation and activation of T and B cells.

TABLE 10 S. Typhimurium vaccine strains with regulated delayedattenuation stimulate higher systemic cytokine production. MouseCytokines concentrations (pg/ml) groups IL-2 IL-4 IL-5 IL-10 IL-12GM-CSF TNF-α BSG  6.5 ± 0.71 13.4 ± 1.27  8.5 ± 1.41  7.8 ± 1.06 15.0 ±0.71 12.5 ± 1.41  9.9 ± 0.14 χ8133  7.3 ± 0.35 18.3 ± 0.35 10.2 ± 1.20 8.5 ± 0.00 17.7 ± 0.92 13.8 ± 1.06 11.3 ± 0.35 (pYA3493) χ8133 ** 12.0± 1.27 27.7 ± 5.44 21.8 ± 3.89 21.7 ± 3.04 42.3 ± 9.55 28.0 ± 2.12 32.3± 3.18 (pYA3634) χ9088  9.5 ± 0.00 27.4 ± 1.56 17.0 ± 0.00 14.0 ± 1.4129.9 ± 3.39 20.9 ± 0.57 18.8 ± 1.77 (pYA3493) χ9088 ** 11.5 ± 0.71 37.3± 1.41 24.5 ± 2.12 22.5 ± 0.71 49.7 ± 1.20 31.7 ± 3.75 31.0 ± 2.12(pYA3634) χ9558 11.8 ± 0.35 34.8 ± 1.06 30.5 ± 2.12 25.8 ± 1.06 53.3 ±1.41 39.2 ± 1.02 37.8 ± 1.06 (pYA3493) χ9558 ** ^(#) 15.0 ± 1.41 46.3 ±1.06 32.0 ± 1.41 26.5 ± 1.48 59.0 ± 8.13 39.8 ± 7.78 39.7 ± 3.32(pYA3634) ** Compared with BSG group of mice all three vaccine strainsstimulate significant higher systemic cytokine production p < 0.01. ^(#)Compared with χ8133(pYA3634) group of mice, χ9558(pYA3634) group of micestimulate significantly higher systemic cytokine production p < 0.05.Evaluation of Protective Immunity.

To examine the ability of Salmonella-rPspA vaccines to protect againstpneumococcal infection, mice were challenged intraperitoneally with5×10⁴ CFU (250 times the LD₅₀) of S. pneumoniae WU2 four weeks afterthey were boosted. Eighty-six percent of the mice immunized withχ9088(pYA3634), seventy-one percent of the mice immunized withχ9558(pYA3634), and twenty-nine percent of the mice immunized withχ8133(pYA3634) were protected from pneumococcal challenge, withstatistical significance (p<0.01). This challenge dose killed 100% ofthe non-immunized, and χ8133(pYA3493), χ9088(pYA3493) and χ9558(pYA3493)immunized mice (Table 11). Following challenge, non-immunized mice, andmice immunized with χ8133(pYA3493) or χ9088(pYA3493) or χ9558(pYA3493)died rapidly.

TABLE 11 Oral immunization with PspA-expressing Salmonella strainsprotects BALB/c mice against i.p. challenge with 5 × 10⁴ CFU of capsulartype 3 S. pneumoniae WU2 Number of Protec- PspA chal- tion expres-lenged rate Vaccine strain^(a) sion^(b) mice Days to death^(c) (%) BSGNA 10 2, 2, 2, 2, 2, 2, 2, 2, 3, 3 0 χ8133(pYA3493) − 10 2, 2, 2, 2, 2,2, 2, 2, 2, 2 0 χ9088(pYA3493) − 10 2, 2, 2, 2, 2, 2, 2, 2, 2, 3 0χ9558(pYA3493) − 10 2, 2, 2, 2, 2, 2, 2, 2, 2, 3 0 χ8133(pYA3634) + 142, 2, 2, 2, 2, 2, 2, 2, 3, 3, 21  3, >15, >15, >15 χ9088(pYA3634) + 142, 3, >15, >15, >15, >15, 86* >15, >15, >15, >15, >15, >15, >15, >15χ9558(pYA3634) + 14 2, 2, 3, 3, >15, >15, >15,  71^(#) >15, >15, >15, >15, >15, >15, >15 ^(a)Mice were orally immunizedtwice at 8-weeks intervals with the indicated vaccine strains. ^(b)+,PspA expressed; −, PspA not expressed; NA, not applicable. ^(c)Fourweeks after the second oral immunization, mice were challenged in twoexperiments with approximately 5 × 10⁴ CFU of S. pneumoniae WU2. Bothexperiments gave similar results, and the data have been pooled forpresentation and analysis. The LD₅₀ of WU2 in nonimmunized BALB/c miceis 2 × 10² (data not shown). *p < 0.001 versus survival of miceimmunized with the χ8133(pYA3634); ^(#) p = 0.001 versus survival ofmice immunized with the χ8133(pYA3634).Passive-Immunization Studies.

A passive-immunization study was conducted to evaluate the roles ofantibody and T-cell mediated immunity afforded by immunization of micewith the recombinant attenuated Salmonella vaccines. One hundredmicroliters of pooled sera, spleen lymphocytes (1×10⁷) or purified CD4+T cells (5×10⁶) taken from immunized mice or controls were administeredby tail vein injection into groups of 5 naïve mice. This was followed 12hours later by intraperitoneal challenge with 5×10⁴ CFU WU2. Micereceiving sera or cells transferred from χ8133(pYA3493), χ9088(pYA3493),χ9558(pYA3493) or BSG immunized mice died with a mean time of 2 days(Table 12). The sera transferred from both χ9088(pYA3634) immunized miceand χ9558(pYA3634) immunized mice protected all 5 naïve mice fromchallenge; while the sera transferred from χ8133(pYA3634) immunized miceprotected 4 mice from challenge, the other mouse died 4 days afterchallenge. Passive transfer of spleen lymphocytes from χ9088(pYA3634)immunized mice protected all 5 naive mice from challenge; spleenlymphocytes from χ9558(pYA3634) immunized mice protected 3 out of 5 micefrom challenge; while the spleen cell transfer from χ8133(pYA3634)immunized mice showed no protection. All the mice receiving CD4+ T cellsfrom any group of immunized mice all died in 2 or 3 days (Table 12).

TABLE 12 Passive transfer of pneumococcal immunity by serum orlymphocytes from donors immunized with PspA Salmonella vaccines %survival of recipients of ^(b) Vaccine strain used to Pooled SpleenPurified CD₄ ⁺ T immunize donors^(a) serum^(c) cells^(d) cells^(e) BSG 00 0 χ8133(pYA3493) 0 0 0 χ9088(pYA3493) 0 0 0 χ9558(pYA3493) 0 0 0χ8133(pYA3634) 80 0 0 χ9088(pYA3634) 100 100*  0 χ9558(pYA3634) 10060^(#) 0 ^(a)Mice were orally immunized at day 0 and boosted 8 weekslater with the indicated vaccine strains. Serum and cells were collected4 weeks after boosting and transferred to groups of 5 naïve mice. ^(b)All recipient mice were challenged by i.p with 5 × 10⁴ CFU WU2 at 12 hafter transfer. Survival was calculated 15 days postchallenge. ^(c)0.1ml of serum intravenously. ^(d)1 × 10⁷ viable spleen cellsintravenously. ^(e)5 × 10⁶ viable CD₄ ⁺ T cells intravenously. *p <0.001, compared to groups of mice receiving passive transfer fromχ8133(pYA3634) immunized or control donors. ^(#)p = 0.001, compared togroups of mice receiving passive transfer from χ8133(pYA3634) immunizedor control donors.Isolation of S. pneumoniae from Blood and Histological Examinations ofChallenged Mice.

Twenty-four hours after intraperitoneal challenge, each mouse was markedand bled. S. pneumoniae was recovered from the blood of mice whichshowed significant signs of weakness and listlessness and died within 7days (6833.3±321.5 CFU/ml), but not from mice that appeared to behealthy and survived past 15 days (p<0.001). Histological analysesshowed severe tissue damage to the lung tissue in mice that died afterchallenge (FIG. 56). Gross visual examination of the lungs of the deadmice from challenge but not the survivors' lungs showed lobar regions ofconsolidation and hemorrhage. Conventional light microscopy ofH&E-stained lung tissue samples of dead mice from challenge demonstratedthat S. pneumoniae caused focal consolidation with extensive mononuclearand polymorphonuclear infiltration and loss of alveolar structure, whichare characteristic of pneumonia.

Discussion of Example 3

Few infectious pathogens can match the global impact of Streptococcuspneumoniae (S. pneumoniae) on illness, complications, sequelae, healthcare costs, and death (28). S. pneumoniae is the most common cause ofcommunity-acquired pneumonia, bacterial meningitis, and acute otitismedia (29, 30). The current 23-valent polysaccharide vaccine has beenshown to prevent pneumococcal pneumonia in immunocompetent young adults,but not in elderly persons (31). A 7-valent conjugated polysaccharidevaccine is licensed for use in children. However, the low serotypecoverage, need for repeated doses, and high price, may decrease theusefulness of the conjugate vaccine, especially in the developing world(32). Developing an inexpensive, safe and effective vaccine against thispathogen is still an urgent demand.

The new regulated delayed attenuation strains χ9088(pYA3634) andχ9558(pYA3634) induced stronger immune responses to PspA as judged byPspA-specific serum antibody levels, PspA specific lymphocytes cytokinesecretion levels, systemic cytokine secretion levels and protection fromvirulent S. pneumoniae challenge. In this example a 10-fold higherchallenge dose of S. pneumoniae WU2 was used compared to earlier studies(13), while the protection rate increased more than twenty percent.

Mixed Th1- and Th2-type immune responses were observed for rPspA. Themechanisms stimulating these types of immune responses by theSalmonella-rPspA vaccine remain unknown. Host defense againstencapsulated bacteria, such as S. pneumoniae, depends on the presence ofopsonic antibodies specific for capsular polysaccharide (33-35).Therefore, antibody levels measured by ELISA may not adequately reflectthe presence of protective antibodies that are capable of triggeringleukocyte effector functions (36). Other investigators have suggestedthat IgG2a is protective during infection with encapsulated bacteria,including pneumococcal infection (36-38). It is possible that underconditions of limiting expression of antibody, in the mouse, IgG2a ishighly effective at fixing complement and promoting opsonophagocytosis(39). It is consistent with our result that after boosting, IgG2abecomes the dominant antibody type. Some investigators reported thatCD4⁺ T cells are very important for the protection against virulent S.pneumoniae challenge (40-41). Our results showed that CD4⁺ T cells fromboth groups of immunized mice did not confer protection. It might bethat for our Salmonella-rPspA vaccines, PspA specific antibodies are themost important factors in protection. T cells are important in helpingthe production of effective antibodies (23, 42, 43), but T cellsthemselves did not have the ability to protect, at least in thisexperiment.

All the control mice challenged intraperitoneally with S. pneumoniae WU2died of septicemia. In surviving mice from the immunized group nobacteria were recovered from blood and also no signs of lung damage weredetected, indicating that these vaccines can protect mice from bothfatal bacteremia and the pneumonia caused by S. pneumoniae.

Although χ9558(pYA3436) possesses the ΔrelA 198::araC P_(BAD) lacl TTdeletion-insertion mutation to provide regulated delayed synthesis ofthe PspA antigen, this feature did not show any benefit to the level ofimmunogenicity over that induced by χ9088 that does not have thisattribute that was shown to be highly beneficial in Example 1. There aretwo probable reasons for this. First, the segment of the Rx1 PspAspecified by pYA3634 is shorter and less stressful on recombinantSalmonella than the Rx1 PspA specified by the codon optimized sequencein pYA4088 used in the studies reported in Example 1. Second, χ9558possesses some 10 genetic alterations and has a genotype almostidentical to S. typhi candidate vaccine strains that will soon beevaluated in human volunteers. This is in contrast to the presence ofonly four genetic alterations in χ9088. The additional mutations inχ9558 are present to render it safe for newborn mice and thus probablyresult in some degree of overattenuation to reduce immunogenicity.Nevertheless, these constructions are important to evaluate since thehistorical fact is that almost all single mutations that render S.typhimurium totally avirulent and highly immunogenic in mice whenintroduced into S. typhi strains and tested in humans are stillpartially virulent and cause disease. It will thus be important toevaluate isogenic strains that only differ by the presence or absence ofthe ΔrelA 198::araC P_(BAD) lacl TT deletion-insertion mutation.Nevertheless, χ9558(pYA3436) was still far superior to χ8133(pYA3634) inregard to immunogenicity and in inducing protective immunity topneumococcal challenge.

In conclusion, the results of this example demonstrated that theSalmonella vaccine strains χ9088(pYA3436) and χ9558(pYA3436) featuringthe novel regulated delayed in vivo attenuation system are superior notonly in inducing PspA specific antibody responses but also in elicitingcellular immunity and cytokine secretion resulting in significantprotection of mice against pneumococcal challenge.

REFERENCES FOR EXAMPLE 3

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Example 4: Design and Construction of S. typhi and S. Paratyphi aVaccine Strains with Regulated Delayed Attenuation and Regulated DelayedSynthesis of Plasmid Encoded Protective Antigens

Attenuated mutants of Salmonella enterica serovar Typhi (S. typhi) andSalmonella enterica serovar Typhimurium (S. typhimurium) have beenextensively studied as multivalent vectors expressing more than 50different bacterial, viral and protozoan antigens in preclinical andclinical trials (1-5). Recombinant S. typhimurium vaccines (RASVs)administered orally can colonize the gut-associated lymphoid tissue(GALT) and the secondary lymphatic tissues, including the liver andspleen, and elicit mucosal, humoral and cellular immune responsesagainst Salmonella and heterologous antigens during infection of themouse (1, 2). There is also good reason to consider use of attenuatedderivatives of S. Paratyphi A as vaccine vectors since infections withthis pathogen causing enteric fever have been increasing in recent yearsdue to the cessation in use of the killed TAB vaccine. This has beencontributed to by immunization against S. typhi by use of the live Ty21Aor conjugate Vi antigen vaccines.

A number of factors may affect the immune response to protectiveantigens including the ability of vaccine strains to invade and colonizethe host GALT, the stability of the plasmid expression system and theantigen sub-cellular location (1, 2, 6). High-level expression ofprotective antigens by RASV strains often impose an energy demand thatdecreases the growth, fitness and ability to colonize lymphoid tissuesresulting in further attenuation and a reduction in immunogenicity (2,7, 8). Means have been developed, such as regulated delayed in vivoantigen synthesis (Example 1), to enhance the ability of vaccine strainsto efficiently invade and colonize GALT after oral immunization (7-10).Another strategy to improve the immune response is to construct strainswith regulated delayed in vivo attenuation such that vaccine strains arebetter able to withstand the stresses and host defence means thatgenerally reduce survival especially of attenuated vaccine strains. Wehave addressed this problem by developing means for regulated delayed invivo attenuations such that vaccine strains are more able to survivethese host induced stresses and behave more like wild-type infectiousSalmonella pathogens at the time of immuniztion. Thus, the combined useof regulated delayed in vivo attenuation and regulated delayed in vivosynthesis of protective antigens both afford means for the RASV to moreefficiently colonize to a higher vaccine cell density effector lymphoidtissues to enhance induction of effective protective immunity.

We have thus constructed S. typhimurium strains to evaluate in mice witha constellation of mutations to not only provide regulated delayed invivo attenuation and regulated delayed in vivo synthesis ofplasmid-specified protective antigens but to also provide inability toestablish persistent carrier states, inability to induce fluid secretionin the intestine, and engender safety for newborns. S. typhimurium UK-1strains with each of the mutations present in some of these strains andnot presented in detail in Examples 1 and 2 are listed in Table 13 alongwith data from infection of female BALB/c mice to indicate thecontributions of these mutations to the level of virulence/attenuation.The suicide vectors used to introduce these mutations into Salmonellastrains are listed in Table 2 and the methods for these constructionsare given in Examples 1 and 2.

TABLE 13 Virulence of S. Typhimurium UK-1 mutants with single deletionor deletion-insertion mutations CFU/ Survival/ Strain Genotype dosetotal χ3761 Wild-type UK-1 4.7 × 10⁸ 0/5 4.7 × 10⁷ 0/5 4.7 × 10⁵ 0/5 4.7× 10⁴ 0/5 4.7 × 10³ 3/5 χ3761 Wild-type UK-1 1.0 × 10⁶ 0/5 1.0 × 10⁵ 0/51.0 × 10⁴ 4/5 1.0 × 10³ 5/5 χ8767 ΔaraBAD23 1.9 × 10⁵ 1/5 UK-1 1.9 × 10⁴1/5 1.9 × 10³ 5/5 χ8477 ΔaraE25 1.1 × 10⁸ 0/4 UK-1 1.1 × 10⁷ 1/4 1.1 ×10⁶ 1/4 1.1 × 10⁵ 2/4 χ8276 ΔasdA16 6.0 × 10⁸ 5/5 UK-1 9.8 × 10⁸ 4/4χ8960 ΔasdA18::TT araC P_(BAD) c2 5.5 × 10⁸ 5/5 UK-1 χ8289 ΔasdA19::TTaraC P_(BAD)c2 1.0 × 10⁹ 4/4 UK-1 χ8958 ΔasdA33 6.4 × 10⁸ 5/5 UK-1 χ8844ΔendA2311 8.6 × 10⁶ 0/4 UK-1 8.6 × 10⁵ 2/4 8.6 × 10⁴ 2/2 χ8844 ΔendA23113.0 × 10⁵ 0/2 UK-1 3.0 × 10⁴ 1/2 χ8989 ΔendA19::araC P_(BAD) lacI TT 1.0× 10⁶ 0/5 UK-1 1.0 × 10⁵ 2/5 1.0 × 10⁴ 2/5 χ8831 Δ(gmd-fcl)-26 5.9 × 10⁵1/4 UK-1 5.9 × 10⁴ 4/4 5.9 × 10³ 4/4 5.9 × 10² 4/4 χ8831 Δ(gmd-fcl)-268.6 × 10⁶ 0/4 UK-1 8.6 × 10⁵ 0/4 8.6 × 10⁴ 0/4 8.6 × 10³ 1/4 χ8650Δpmi-2426 1.7 × 10⁹ 8/8 UK-1 1.7 × 10⁸ 8/8 Nutrient Broth 1.7 × 10⁷ 7/81.7 × 10⁶ 4/4 1.7 × 10⁵ 4/4 χ8650 Δpmi-2426 1.5 × 10⁹ 3/8 UK-1 1.5 × 10⁸7/8 Nutrient Broth + 1.5 × 10⁷ 7/8 0.5% mannose + 1.5 × 10⁶ 4/4 0.5%glucose 1.5 × 10⁵ 4/4 χ8882 ΔrelA1123 8.0 × 10⁷ 0/4 UK-1 8.0 × 10⁶ 1/58.0 × 10⁵ 1/4 8.0 × 10⁴ 3/4 8.0 × 10³ 4/4 χ8990 ΔrelA196::araC P_(BAD)lacI TT 1.0 × 10⁵ 1/5 UK-1 1.0 × 10⁴ 4/5 1.0 × 10³ 5/5 χ8923 ΔsopB19251.2 × 10⁷ 1/5 UK-1 1.2 × 10⁶ 2/5 1.2 × 10⁵ 5/5 1.2 × 10⁴ 5/5

Strain χ9558 Δpmi-2426 Δ(gmd-fcl)-26 ΔP_(fur81)::TT araC P_(BAD) furΔP_(crp527)::TT araC P_(BAD) crp ΔasdA27::TT araC P_(BAD) c2 ΔaraE25ΔaraBAD23 ΔrelA 198::araC P_(BAD) lacl TT ΔsopB1925 ΔagfBAC811 was usedin Example 3 to deliver heterologous antigens such as the S. pneumoniaeα-helical domain of the Rx1 PspA antigen encoded by pYA3634 eithersecreted into the extra-cellular environment or displayed on the vaccinecarrier surface. Such approaches are based on observations that antigenslocalized on the surface of Salmonella cells or extracellularly secretedproduce greater immune responses and protection (6, 11-13). In addition,secretion of heterologous antigens by RASV may decrease the toxicity ofthe protein to the bacterial vector, facilitate bacterial growth andantigen uptake by antigen-presenting cells (APC) and continuouslystimulate the host immune system during the colonization of lymphatictissues by Salmonella, so as to enhance the immune response against theheterologous antigens (5, 14).

Streptococcus pneumoniae, a gram-positive human pathogen, causes serioushealth problems, including community-acquired pneumonia, otitis media,meningitis, and bacteremia in persons of all ages. S. pneumoniae is aleading agent of childhood pneumonia worldwide, resulting in about 3million deaths per year (15). The pneumococcal surface protein A (PspA)and pneumococcal surface protein C (PspC) have been considered aspneumococcal subunit vaccine candidates. PspA and PspC/Hic are expressedin all clinically isolated pneumococcal strains (16-18). Immuneresponses to PspA and PspC can protect mice against virulent S.pneumoniae challenge (6, 17, 19-23).

Toward this end of making a suitable vaccine to prevent pneumococcaldisease in newborns and infants, we have introduced the improved Asd⁺vector pYA4088 (Example 1) that expresses at high level the longer Rx1PspA specified by a codon-optimized sequence into χ9558. In addition torepeating the immunogenicity and protection studies described in Example3 with superior results, we have demonstrated that this recombinantstrain can be orally administered to newborn mice only a few hours oldat doses of 10⁸ CFU or more with no deaths, disease symptoms orimpairment in growth. Furthermore, when the χ9558(pYA4088) strain isinoculated intranasally into six-week old BALB/c mice, there is nocolonization of the olfactory lobe or brain and no development ofmeningitis as found after intranasal inoculation of other Salmonellastrains.

Based on the above successes, we have generated S. typhi vaccine vectorsas listed in Table 1 that have most all of the mutations present inχ9558. Since there has been a lack of success of recombinant attenuatedS. typhi vaccines derived from the widely used Ty2 strain, we havegenerated recombinant strains that have the rpoS mutation in the Ty2strain replaced with the wild-type rpoS allele and have also derived avaccine strain from the Chilean clinical isolate ISP1820.1 χ9639 is theS. typhi Ty2 derived strain that is RpoS⁻ and has the genotypeΔP_(crp527)::TT araC P_(BAD) crp ΔP_(fur81)::TT araC P_(BAD) furΔpmi-2426 Δ(gmd-fcl)-26 ΔsopB1925 ΔrelA 198::araC P_(BAD) lacl TTΔaraE25ΔtviABCDE10 ΔagfBAC811 ΔasdA33 and χ9640 is the Ty2 RpoS⁺derivative with the identical genotype as in χ9639. χ9633 is the S.typhi ISP1820 derivative is RpoS⁺ and has the genotype ΔP_(crp527)::TTaraC P_(BAD) crp ΔP_(fur81)::TT araC P_(BAD) fur Δpmi-2426 Δ(gmd-fcl)-26ΔsopB1925 ΔrelA 198::araC P_(BAD) lac TT ΔaraE25 ΔaraBAD23 ΔtviABCDE10ΔagfBAC811 ΔasdA33. χ9639 and χ9640 do not have the ΔaraBAD23 mutationpresent in χ9633 since the parental Ty2 strain is already unable to usearabinose for growth or as a fermentation substrate. All three strainspossess the attenuating ΔtviABCDE10 mutation that eliminates synthesisof the capsular Vi antigen that is unique to S. typhi strains and is notmade by S. typhimurium. The Δ(gmd-fcl)-26 and ΔagfBAC811 mutations blocksynthesis of colanic acid and thin aggregative fimbriae (curli),respectively, that prevent formation of biofilms and preclude persistantcolonization of gallstones in the gall bladder. The ΔsopB1925 mutationreduces the potential to induce fluid secretion in the intestinal trackand also eliminates a means by which Salmonella suppresses induction ofimmune responses. Thus, this mutation increases the immunogenicity ofthese S. typhi antigen delivery vaccine vectors.

We have also designed and constructed vaccine vector derivatives in S.Paratyphi A and note that χ9857 (Table 1) with the genotypeΔP_(crp527)::TT araC P_(BAD) crp ΔP_(fur81)::TT araC P_(BAD) furΔpmi-2426 Δ(gmd-fcl)-26 ΔagfBAC811 ΔrelA 198::araC P_(BAD) lacl TTΔsopB1925 ΔaraE25ΔaraBAD23 ΔasdA33 has much the samr properties as χ9558and the three S. typhi vaccine vectors described above.

REFERENCES FOR EXAMPLE 4

-   1. Curtiss R. III (2005) in Mucosal Immunology, ed. J. Mestecky M E    L, W. Strober, J. Bienenstock, J. R. McGhee, and L. Mayer (ed)    (Academic Press, San Diego).-   2. Curtiss R, III, Zhang X, Wanda S Y, Kang H Y, Konjufca V, Li Y,    Gunn B, Wang S, Scarpellini G, & Lee. I S (2007) in Virulence    Mechanisms of Bacterial Pathogens, ed. K. A. Brogden F C M, N.    Cornick, T. B. Stanton, Q. Zhang, L. K. Nolan, and M. J.    Wannemuehler (ASM Press, Washington D.C.), pp. 297-313.-   3. Curtiss R, III, Kelly S M, & Hassan J O (1993) Live oral    avirulent Salmonella vaccines. Vet. Microbiol. 37: 397-405.-   4. Doggett T A & Curtiss R, III (1992) Delivery of antigens by    recombinant avirulent Salmonella strains. Adv. Exp. Med. Biol. 327:    165-173.-   5. Spreng S, Dietrich G, & Weidinger G (2006) Rational design of    Salmonella-based vaccination strategies. Methods 38: 133-143.-   6. Kang H Y & Curtiss R, III, (2003) Immune responses dependent on    antigen location in recombinant attenuated Salmonella typhimurium    vaccines following oral immunization. FEMS Immunol. Med. Microbiol.    37: 99-104.-   7. Chatfield S N, Charles I G, Makoff A J, Oxer M D, Dougan G,    Pickard D, Slater D, & Fairweather N F (1992) Use of the nirB    promoter to direct the stable expression of heterologous antigens in    Salmonella oral vaccine strains: development of a single-dose oral    tetanus vaccine. Biotechnology (N Y) 10: 888-892.-   8. Roberts M, Li J, Bacon A, & Chatfield S (1998) Oral vaccination    against tetanus: comparison of the immunogenicities of Salmonella    strains expressing fragment C from the nirB and htrA promoters.    Infect. Immun. 66: 3080-3087.-   9. Curtiss R, III, S-Y. Wanda, X. Zhang, B. Gunn. (2007) Salmonella    vaccine vectors displaying regulated delayed in vivo attenuation to    enhance immunogenicity, abstr. E-061, p 278. Abstr. 107th Gen. Meet.    Am. Soc. Microbiol. American Society for Microbiology, Washington,    D.C.-   10. Wang S, Y. Li, G. Scarpellini, W. Kong, Curtiss R, III (2007)    Salmonella vaccine vectors displaying regulated delayed antigen    expression in vivo to enhance immunogenicity, Abstr. E-064, p 278.    Abstr. 107th Gen. Meet. Am. Soc. Microbiol. American Society for    Microbiology, Washington, D.C.-   11. Kaufmann S H & Hess J (1999) Impact of intracellular location of    and antigen display by intracellular bacteria: implications for    vaccine development. Immunol. Lett. 65: 81-84.-   12. Hess J, Gentschev I, Miko D, Welzel M, Ladel C, Goebel W, &    Kaufmann S H (1996) Superior efficacy of secreted over somatic    antigen display in recombinant Salmonella vaccine induced protection    against listeriosis. Proc. Natl. Acad. Sci. USA 93: 1458-1463.-   13. Hess J, Grode L, Gentschev I, Fensterle J, Dietrich G, Goebel W,    & Kaufmann S H (2000) Secretion of different listeriolysin cognates    by recombinant attenuated Salmonella typhimurium: superior efficacy    of haemolytic over non-haemolytic constructs after oral vaccination.    Microbes Infect. 2: 1799-1806.-   14. Gentschev I, Glaser I, Goebel W, McKeever D J, Musoke A, &    Heussler V T (1998) Delivery of the p67 sporozoite antigen of    Theileria parva by using recombinant Salmonella dublin: secretion of    the product enhances specific antibody responses in cattle. Infect.    Immun. 66: 2060-2064.-   15. Greenwood B (1999) The epidemiology of pneumococcal infection in    children in the developing world. Philos. Trans. R. Soc. Lond. B.    Biol. Sci. 354: 777-785.-   16. Iannelli F, Oggioni M R, & Pozzi G (2002) Allelic variation in    the highly polymorphic locus pspC of Streptococcus pneumoniae. Gene    284: 63-71.-   17. Brooks-Walter A, Briles D E, & Hollingshead S K (1999) The pspC    gene of Streptococcus pneumoniae encodes a polymorphic protein,    PspC, which elicits cross-reactive antibodies to PspA and provides    immunity to pneumococcal bacteremia. Infect. Immun. 67: 6533-6542.-   18. Hollingshead S K, Becker R, & Briles D E (2000) Diversity of    PspA: mosaic genes and evidence for past recombination in    Streptococcus pneumoniae. Infect. Immun. 68: 5889-5900.-   19. Briles D E, Hollingshead S K, King J, Swift A, Braun P A, Park M    K, Ferguson L M, Nahm M H, & Nabors G S (2000) Immunization of    humans with recombinant pneumococcal surface protein A (rPspA)    elicits antibodies that passively protect mice from fatal infection    with Streptococcus pneumoniae bearing heterologous PspA. J. Infect.    Dis. 182: 1694-1701.-   20. Briles D E, Hollingshead S K, Nabors G S, Paton J C, &    Brooks-Walter A (2000) The potential for using protein vaccines to    protect against otitis media caused by Streptococcus pneumoniae.    Vaccine 19 Suppl 1: S87-S95.-   21. Briles D E, King J D, Gray M A, McDaniel L S, Swiatlo E, &    Benton K A (1996) PspA, a protection-eliciting pneumococcal protein:    immunogenicity of isolated native PspA in mice. Vaccine 14: 858-867.-   22. Kang H Y, Srinivasan J, & Curtiss R, III, (2002) Immune    responses to recombinant pneumococcal PspA antigen delivered by live    attenuated Salmonella enterica serovar Typhimurium vaccine. Infect.    Immun. 70: 1739-1749.-   23. Nayak A R, Tinge S A, Tart R C, McDaniel L S, Briles D E, &    Curtiss R, III, (1998) A live recombinant avirulent oral Salmonella    vaccine expressing pneumococcal surface protein A induces protective    responses against Streptococcus pneumoniae. Infect. Immun. 66:    3744-3751.

Example 5: Biological Containment

Live attenuated pathogens such as Salmonella enterica have beendeveloped as homologous vaccines to protect against Salmonellainfections and as carriers of heterologous antigens because of theircapacity for efficient mucosal antigen delivery (1, 2). A variety ofattenuating mutations and antibiotic-free balanced-lethal plasmidstabilization systems has been developed for this purpose (1, 3-5).However, biological containment systems are required to address thepotential risk posed by the unintentional release of these geneticallymodified organisms into the environment, a subject of considerableconcern (6, 7). Such release can lead to unintentional immunizations andthe possible transfer of cloned genes that might represent virulenceattributes in some cases. A number of mutations have been identified inS. typhimurium, including shdA, misL and ratB, that reduce environmentalshedding in mice without negatively influencing immunogenicity (8).While these mutations lead to a reduction in fecal shedding, it is notclear how long these strains will persist in the environment. Therefore,more effective systems need to be developed. Our approach has been todevelop a biological containment system that will allow the vaccinestrain time to colonize the host lymphoid tissues, a requirement forinducing a robust immune response (9, 10) and eventually lead to celldeath by lysis, thus preventing spread of the vaccine strain into theenvironment.

The intracellular location of antigens in a recombinant attenuatedSalmonella vaccine (RASV) can significantly influence the level ofinduced immune response upon immunization (11). Thus, if the antigen isretained in the cytoplasm and must be released by the actions of theimmunized host, the immune response to the antigen is not as strong aswhen the antigen is secreted (11, 12). We hypothesize that the releaseof an expressed antigen by a RASV delivery strain within the lymphoidtissues of an immunized animal by programmed lysis would further enhancethe immune response to the expressed antigen.

In this example, a RASV programmed bacterial lysis system vectoring theβ-lactamase-PspA fusion protein was constructed. It was previously shownthat this fusion protein is directed to the periplasm, but only about 10to 20% is released into the extracellular environment (13). This newsystem combines the features of a previously described antigenexpression plasmid with a novel programmed bacterial lysis systemdesigned to release antigen into the host tissues to induce anefficacious immune response and to provide biological containment of theRASV.

Materials and Methods for Example 5

Bacterial Strains, Plasmids, Media, and Growth Conditions:

Bacterial strains and plasmids used in this example are listed in Tables1 and 2, respectively. S. typhimurium strains with asdA gene deletionswere grown at 37° C. in LB broth or on LB agar (14) supplemented with 50g/ml DAP (3). Transformants containing araC P_(BAD) asdA murA plasmidswere selected on LB agar plates containing 0.2% arabinose. We used 0.02%arabinose in LB broth cultures to prevent pH changes in the mediumcaused by metabolism of arabinose that may affect the physiology of thebacterial cells. LB agar containing 5% sucrose, no sodium chloride, wasused for sacB gene-based counterselection in allelic exchangeexperiments (15). For mouse inoculation, Salmonella strains were grownwith aeration after inoculated with a 1/20 dilution of a non-aeratedstatic overnight culture.

Strain Characterization:

Vaccine strains were compared with vector controls for stability ofplasmid maintenance, arabinose-dependent growth and antigen synthesis.Molecular genetic attributes were confirmed by PCR with appropriateprimers. Lipopolysaccharide (LPS) profiles of Salmonella strains wereexamined by described methods (16). For detection of PspA in RASV, 12 μlor 2 μl of cultures at an OD₆₀₀ of 0.8 were subjected to SDS-PAGE orimmunoblot analysis, respectively.

Construction of a Regulated Programmed Lysis S. typhimurium VaccineHost-Vector System:

The ΔP_(murA7)::araC P_(BAD) murA deletion-insertion mutation wasconstructed by standard methods and introduced into wild-type strainχ3761 to yield χ8645. The ΔasdA19::araC P_(BAD) c2 deletion-insertionmutation was introduced into χ8645 by P22HT int transduction from χ8290(ΔasdA19::TT araC P_(BAD) c2). As described in Example 4 and presentedin Table 13, these two types of mutations render Salmonella totallyavirulent. However, as noted in the Background to the Invention, theΔP_(murA7)::araC P_(BAD) murA deletion-insertion mutation represents anexample of a mutation conferring a regulated delayed attenuationphenotype since a strain with this mutation would be expected to growand divide a generation or two before undergoing muramic acid-less deathby lysis. The ΔendA2311, Δ(gmd-fcl)-26 and ΔrelA 1123 mutations wereadded sequentially using suicide vectors (see Table 2) resulting invaccine strain χ8937. The presence of mutations and absence of suicidevector sequences were confirmed by PCR using suitable primer sets (Table14). The primers and steps used to construct plasmids are described asfollows. The E. coli B/r araC P_(BAD) activator-promoter was derivedfrom pBAD18 (17). The E. coli K-12 araC P_(BAD) activator-promoter wasPCR amplified using primers araC-NsiI and EaraBAD-EcoRI from strainχ289. The SD-GTG mutation in pYA3530 was introduced into the asdA geneby PCR. The ATG-murA gene was amplified by PCR from E. coli K-12 strainχ289 (glnV42 λ⁻ T3^(r)) using primers EmurA-EcoRI 5′ and EmurA-EcoRI 3′,then the ATG start codon of the murA gene was changed to GTG by PCR. TheP22 P_(R) promoter was derived from plasmid pMEG104. TheP_(trc)-5ST1T2-P_(BR) ori fragment came from plasmid pYA3342. Thefragment including in-frame fusion of the rPspA Rx1 (α-helical region ofPspA from amino acid residue 3 to 257 of mature PspA_(Rx1)) to theβ-lactamase signal sequence was derived from pYA3634 (18). Expression ofthe rPspA Rx1 antigen was verified by SDS-PAGE and western blot analysiswith the anti-PspA monoclonal antibody Xi126 (19).

TABLE 14 Primers used in this example Primer Name SequenceA. Construction of plasmid pYA3681 GTG asd  GTG asd 5'cag gaa aaa aac gct gtg aaa aat gtt gg GTG asd 3'gtc ctt ttt ttg cga cac ttt tta caa cc araC P_(BAD) GTG asd araC-Smalcga ccc ggg atc gat ctg tgc ggt att tca cac cg asd-Smalgca ccc ggg tcg aca gat cct tgg cgg cga gaa ag araC* P_(BAD)(from χ289)EaraC-Nsil cca atg cat aat gtg cct gtc aaa tgg EaraBAD-EcoRIcgg aat tcg cta gcc caa aaa aac g MurA EmurA-EcoRI 5'cgg aat tct gag aac aaa cta aat gg EmurA-EcoRI 3'cgg aat tct tat tcg cct ttc aca cgc GTG murA EMGTGRV-Ncolcat gcc atg gag ctc ggt acc cgg gga t EMGTG-Ncol-EcoRIcat gcc atg gaa ttc tga gaa caa act aag tgg ata  aat ttc gtg ttc agP_(trc)-pBR ori cassette P_(trc)-Pmelagc ttt gtt taa acg gat ctt ccg gaa gac ctt cca ttc Xbal-pBRgct cta gac tgt cag acc aag ttt act cat a Synthetic rrfG TT rrfG TTaac tgc agt cta gat tat gcg aaa ggc cat cct gac gga tgg cct ttt tgt tta aac gga tcc gc B. Construction of plasmid pYA3685Ncol-bla-PspA cat gcc atg ggt att caa cat ttc cgt gtc gcc ctt att cSmal-TAA-PspA tcc ccc ggg cta tta ttc tac att att gtt ttc TC. Construction of suicide vectors ΔrelA1123 relA C-SphIaca tgc atg ccc aga tat ttt cca gat ctt cac relA C-EcoRIcgg aat tca ccc cag aca gta atc atg tag cgg relA N-EcoRIcgg aat tca agg gac cag gcc tac cga ag relA N-BamHIcgg gat ccg agg gcg ttc cgg cgc tgg tag aa Δ(gmd-fcl)-26 wcaF-XbaIgct cta gat cct caa ata gtc ccg tta gg wcaF-SmaItcc ccc ggg caa aat att gta tcg ctg g gmm-SphIgcacgc atg ctc agg cag gcg taa atc gct ct gmm-XbaIcct cta gac aat gtt ttt acg tca gga aga tt ΔendA2311 endAN-BamHIcgg gat ccg cta cga aat ccg cct caa c endAN-HindIIIccc aag ctt agc aaa acg agc ccg caa cg endAC-HindIIIccc aag ctt cct aca cta gcg gga ttc ttg endAC-SphIaca tgc atg ccg cag cgc tca gag

Strain Characterization:

Vaccine strains were compared with vector controls for stability ofplasmid maintenance, arabinose-dependent growth and antigen synthesis.Molecular genetic attributes were confirmed by PCR with appropriateprimers. Lipopolysaccharide (LPS) profiles of Salmonella strains wereexamined by described methods (16). For detection of PspA in RASV, 12 μlor 2 μl of cultures at an OD₆₀₀ of 0.8 were subjected to SDS-PAGE orimmunoblot analysis, respectively.

Examination of Cell Lysis In Vitro:

Overnight cultures of strains were grown in LB broth supplemented with0.002% arabinose. We used 0.002% arabinose to prevent the accumulationof arabinose within bacterial cells to allow us to detect cell lysisduring the short time frame used for this experiment. Cultures werediluted 1:400 into fresh pre-warmed LB broth supplemented with orwithout 0.02% arabinose 3-galactosidase activity in supernatant andcell-pellet fractions were assayed at indicated time points as described(20).

Colonization of Mice with the Regulated Programmed Lysis SalmonellaVaccine Strain:

Seven-week-old female BALB/c mice (3 mice for each time point) weredeprived of food and water for 4 h before oral administration ofSalmonella vaccine strains. These strains were grown with aeration in LBbroth supplemented with 0.02% arabinose to an optical density at 600 nm(OD₆₀₀) of 0.85 from a non-aerated static overnight culture. 1.3×10⁹ CFUof χ8937(pYA3681) in 20 μl of phosphate-buffered saline containing 0.01%gelatin (BSG) was orally administered to the mice at the back of themouth with a pipette tip. Food and water were returned to the animals 30to 45 min later. Mice were euthanized at indicated times and theirPeyer's patches, spleens, and livers were collected aseptically. Tissueswere homogenized and plated on LB agar with 0.2% arabinose to evaluatecolonization and persistence, and onto LB agar plates without arabinoseto screen for arabinose-independent mutants.

Immunization of Mice:

Groups of 5 seven-week-old female BALB/c mice were orally vaccinatedwith either 1.3×10⁹ CFU S. typhimurium vaccine strain χ8937(pYA3685)(expressing rPspA Rx1) or 1.1×10⁹ CFU host-vector controlsχ8937(pYA3681) as described above. A second oral dose of 1.2×10⁹ CFUχ8937(pYA3685) or 1.1×10⁹ CFU χ8937(pYA3681) was given one week later.The immunized mice were monitored for 60 days for evidence of illness byobserving them daily for evidence of diarrhea, ruffled (ungroomed) fur,or irritability. None of these symptoms of infection were observed inany of the mice. Blood was collected at weeks 2, 4, 6, 8 afterimmunization. Serum fractions were stored at −20° C. Vaginal secretionspecimens were collected by wash with 50 μl BSG, solid material wasremoved by centrifugation and secretion samples were stored at −20° C.(21).

Antigen

Preparation: rPspA Rx1 protein and S. typhimurium SOMPs were purified asdescribed (13).

Enzyme Linked Immunosorbent Assay (ELISA):

The procedures used for detection of antibody have been describedelsewhere (13, 22). Briefly, polystyrene 96-well flat-bottom microtiterplates (Nunc, Roskilde, Denmark) were coated with S. typhimurium SOMPs(100 ng/well) or purified rPspA Rx1 (100 ng/well). Antigens suspended insodium carbonate-bicarbonate coating buffer (pH 9.6) were applied with100 μl volumes in each well. Vaginal secretions obtained from the sameexperimental group were pooled and diluted 1:10, and sera were diluted1:1280 for detection of IgG and 1:400 for IgG1 and IgG2a, respectively.A 100 μl volume of diluted sample was added to individual wells induplicate. Plates were treated with biotinylated goat anti-mouse IgG,IgG1, or IgG2a (Southern Biotechnology Inc., Birmingham) for sera andIgA for vaginal secretions.

Statistical Analysis:

Most data were expressed as means±standard error. The means wereevaluated with One-way ANOVA and LSD tests for multiple comparisonsamong groups. p<0.05 was considered statistically significant.

Construction of a Regulated Programmed Lysis System.

Diaminopimelic acid (DAP) and muramic acid are essential components ofthe peptidoglycan layer of the bacterial cell wall (23). The asdA geneencodes an enzyme essential for DAP synthesis and the murA gene encodesthe first enzyme in muramic acid synthesis (24, 25). To test thefeasibility of an arabinose-regulated asdA-based lysis system, weintroduced the ΔasdA16 deletion mutation into S. typhimurium UK-1resulting in strain χ8276. We then introduced plasmid pYA3450, whichcarries the asdA gene with an ATG start codon transcribed from theP_(BAD) promoter which is activated by the AraC protein in the presenceof arabinose (26), into χ8276 to yield χ8276(pYA3450). However, we foundthat growth of this strain was not arabinose-dependent, indicating thatthe level of residual transcription from the P_(BAD) promoter in theabsence of arabinose was sufficient to produce enough Asd for growth. Inaddition, χ8276(pYA3450) also retained wild-type virulence in mice. Toreduce translational efficiency, we changed the asdA start codon fromATG to GTG, generating plasmid pYA3530. The GTG start codonsignificantly decreased the level of Asd expression (FIG. 37A). However,strain χ8276(pYA3530) still exhibited arabinose-independent growth anddid not lyse in media devoid of arabinose. This problem was overcome byadditional modifications described below, including addition of the murAgene.

Unlike lethal asdA deletions, which can be overcome by the addition ofDAP to the growth medium, murA deletions, also lethal, cannot beovercome by nutritional supplements. Therefore, a conditional-lethalmurA mutation was created by replacing the chromosomal murA promoterwith the araC P_(BAD) activator-promoter. We introduced theΔP_(murA7)::araC P_(BAD) murA mutation into wild-type S. typhimuriumresulting in strain χ8645. To evaluate the predicted arabinose-dependentmurA transcription, the strain was inoculated with and without arabinoseinto several media containing nutritional components that are likely tobe encountered by a vaccine strain, including 1% rodent chow, 1% chickenfeed or 1% chicken breast meat in minimal medium (27). As expected,growth was not only dependent on the presence of arabinose, butbacterial titers dropped in the absence of arabinose, indicative of celllysis (FIG. 37B). These results confirm that the ΔP_(murA7)::araCP_(BAD) murA mutation confers a regulated delayed attenuation phenotypesince some growth was possible prior to onset of death by lysis.

We combined the asdA and murA systems, providing redundant mechanisms toensure cell death. However, as described above, we first needed toreduce the amount of Asd produced from our plasmid. The araC P_(BAD)promoter-activator we used for all the previously described constructswas derived from an E. coli B/r strain (17). We discovered that when wesubstituted the araC P_(BAD) promoter-activator from E. coli K-12 strainχ289, transcription from the plasmid was more tightly regulated andarabinose-dependent growth was achieved. We then inserted a murA gene inbetween the P_(BAD) promoter and the asdA gene to further decrease thetranscription level of asdA. Finally, we introduced P22 P_(R), aC2-regulated promoter, with opposite polarity at the 3′ end of the asdgene to interfere with transcription of the plasmid asdA and murA genesand to direct synthesis of antisense mRNA to block translation of mRNAtranscribed from these genes during programmed lysis when arabinose isabsent. Transcription terminators (TT) flank all plasmid domains so thatexpression in one domain does not affect the transcriptional activitiesof any other domain. The resulting plasmid was designated pYA3681 (FIG.14).

The host strain for this system was constructed by introducing a ΔasdAmutation into the ΔP_(murA7)::araC P_(BAD) murA mutant strain χ8645. Tofacilitate regulation of P22 P_(R) in plasmid pYA3681, we introduced theΔasdA19 deletion/insertion mutation, in which the P22 phage C2 repressorgene under transcriptional control of the P_(BAD) promoter was insertedinto the ΔasdA16 deletion (FIG. 38A). Three additional mutationsdesigned to enhance lysis and facilitate antigen delivery were alsoincluded in this strain (FIG. 38A). The Δ(gmd-fcl)-26 mutation deletesgenes encoding enzymes for GDP-fucose synthesis, thereby precluding theformation of colanic acid, a polysaccharide made in response to stressassociated with cell wall damage (28). This mutation was includedbecause we have observed that under some conditions, asdA mutants cansurvive if they produce copious amounts of colanic acid (29). Therefore,by deleting the genes required for colanic acid synthesis, we circumventthis possibility. The ΔrelA 1123 mutation uncouples cell wall-less deathfrom dependence on protein synthesis to further ensure that the bacteriado not survive in vivo or after excretion and to allow for maximumantigen production in the face of amino acid starvation resulting from alack of aspartate semi-aldehyde synthesis due to the asdA mutation (30,31). This regulated lysis system S. typhimurium strain also haspotential for use as a DNA vaccine delivery vector. Therefore, weincluded a ΔendA mutation which eliminates the periplasmic endonucleaseI enzyme (32), to increase plasmid survival upon its release into thehost cell. The resulting strain, χ8937 (ΔasdA19::araC P_(BAD) c2ΔP_(murA7)::araC P_(BAD) murA Δ(gmd-fcl)-26 ΔrelA 1123 ΔendA2311),requires both arabinose and DAP for growth (FIG. 38B).

pYA3681 was introduced into S. typhimurium χ8937. Growth of theresulting strain χ8937(pYA3681) required arabinose (FIG. 38B). Theplasmid was stably maintained for 50 or more generations when grown inthe presence of arabinose and DAP. In the presence of arabinose, theplasmid-encoded copies of asdA and murA and the chromosomally encodedcopies of murA and c2 are transcribed from their respective P_(BAD)promoters, allowing for bacterial growth and repression of the P22 P_(R)promoter by C2 (FIG. 39). In the absence of arabinose, the P_(BAD)promoters cease to be active, with no further synthesis of Asd and MurAor C2. The concentrations of Asd, MurA and C2 decrease due to celldivision. The decreased concentration of Asd and MurA leads to reducedsynthesis of DAP and muramic acid and imbalanced synthesis of thepeptidoglycan layer of the cell wall. As the C2 concentration drops, P22P_(R) is derepressed resulting in P_(R)-directed synthesis of anti-sensemRNA which blocks translation of residual asdA and murA mRNA. Theseconcerted activities lead to cell lysis.

Regulated Programmed Lysis and Biological Containment Properties afterColonization of Lymphoid Tissues.

The regulated lysis vaccine strain χ8937(pYA3681) grew well in LB brothsupplemented with 0.02% arabinose, but began to die after one hour ofincubation in LB broth without arabinose (FIG. 40A). To evaluate celllysis, release of the cytoplasmic enzyme β-galactosidase into culturesupernatants was used as an indicator. The atrBI3::MudJ allele whichdirects constitutive expression of β-galactosidase (13) was transducedinto S. typhimurium wild-type as a non-lysis control and into vaccinestrain χ8937, resulting in strains χ9379 and χ9380, respectively. Wethen introduced plasmid pYA3681 into χ9380 to yield χ9380(pYA3681). Theratio of β-galactosidase activity in the supernatant (releasedβ-galactosidase) or cell pellet (retained cell-associated3-galactosidase) versus total β-galactosidase activity (supernatant pluscell pellet) indicated the extent of cell lysis. Release ofβ-galactosidase by strain χ9380(pYA3681) occurred only in medium lackingarabinose (FIG. 40B). Conversely, the amount of cell-associatedβ-galactosidase decreased over time when χ9380(pYA3681) was grown inmedium without arabinose, but no decrease was seen in media containingarabinose. β-galactosidase release was not observed when the wild-typecontrol strain χ9379 was grown without arabinose. These results areconsistent with our expectations for the arabinose-regulated cell lysisphenotype.

To evaluate virulence, BALB/c mice were orally inoculated with doses inexcess of 10⁹ CFU of the host-vector strain χ8937(pYA3681), a dose50,000 times the LD₅₀ of the wild-type parent strain, χ3761. During the30 days observation period after dosing, we observed no deaths or signsof illness in any of the mice. Colonization by strain χ8937(pYA3681) wasevaluated in eight-week old female mice orally inoculated with 10⁹ CFU.The strain transiently colonized lymphoid tissues (FIG. 40C) and nobacteria were recovered by four weeks post-inoculation. Noarabinose-independent Salmonella mutants were recovered at any timeduring this experiment. These results indicate that a wild-typeSalmonella strain engineered with this programmed lysis system isattenuated and is efficiently cleared from the host followingcolonization of lymphoid tissues.

Construction of the rPspA Rx1-expressing Plasmid.

It was previously shown that a recombinant protein fusing the first 35amino acids of β-lactamase to the α-helical region of PspA (rPspA Rx1)is highly immunogenic when delivered by a recombinant avirulent S.typhimurium (13). We utilized a similar fusion to evaluate the abilityof our regulated lysis strain to deliver an antigen to host tissues. ADNA fragment encoding the in-frame fusion of the 3-lactamase leadersequence from plasmid pBR322 to rPspA Rx1 (α-helical region of PspA fromamino acid residue 3 to 257 of mature PspA_(Rx1)) was inserted intopYA3681 to yield pYA3685 (FIG. 41A). The nucleic acid encoding theantigen is constitutively expressed from the P_(trc) promoter.Production of rPspA Rx1 in S. typhimurium χ8937(pYA3685) grown in mediawith arabinose was detected by western blot analysis with the anti-PspAmonoclonal antibody (FIG. 41B), and we confirmed that the fraction ofantigen secreted to the periplasm was similar to that reportedpreviously (13). The strain did not grow on LB agar without arabinoseand expression of the recombinant antigen did not interfere withprogrammed lysis when χ8937(pYA3685) was grown in LB broth withoutarabinose (FIG. 41C).

Immune Responses in Mice after Oral Immunization with the RegulatedProgrammed Lysis Host-Vector System.

The antibody responses to Salmonella outer membrane proteins (SOMPs) andto the foreign antigen rPspA Rx1 in sera and vaginal secretions of theimmunized mice were measured (FIG. 42). The maximum serum IgG responseto PspA was observed at 6 weeks and responses at all time points weresignificantly greater than in the control group, where no response wasdetected (p<0.05)(FIG. 42A). The anti-SOMP IgG response was slower todevelop in mice vaccinated with χ8937(pYA3685) than in the controlgroup, with significant differences between groups at weeks 2 and 6(p<0.05). This could be a result of differences in the ability of thetwo strains to survive systemically brought about by the antigen load inχ8937(pYA3685). However, both χ8937(pYA3681) and χ8937(pYA3685) elicitedequivalent anti-SOMP IgA responses in vaginal secretions, with nosignificant differences between groups after two weeks, while rPspARx1-specific IgA was detected only in samples from mice immunized withvaccine strain χ8937(pYA3685) (p<0.05) (FIG. 42B).

IgG Isotype Analyses

The type of immune responses to SOMPs and the rPspA Rx1 were furtherexamined by measuring the levels of IgG isotype subclasses IgG2a andIgG1 (FIG. 43). The Th1-helper cells direct cell-mediated immunity andpromote class switching to IgG2a, and Th2 cells provide potent help forB-cell antibody production and promote class switching to IgG1 (33).IgG2a isotype dominant responses were observed for the SOMP antigensindicating that the vaccine induced a strong cellular immune responseagainst Salmonella. In contrast to the strong Th1 responses to SOMPs, aTh1- and Th2-type mixed response was observed for the rPspA Rx1 antigen(FIG. 43).

Discussion of Example 5

Our long-term goals are to develop RASVs for oral administration,protecting humans and animals against a variety of mucosal pathogens.Immunization with live Salmonella vaccines introduces the potential forrelease of the bacteria into the environment, possibly leading tounintended immunizations. The objective of this example was to constructand evaluate a biological containment system that would be consistentwith the requirements for efficacious vaccination, in particular,colonization of host lymphoid tissues for an amount of time sufficientfor optimal antigen delivery. RASV are capable of delivering a varietyof bacterial, viral, fungal, and parasitic antigens thereby elicitinghumoral and cellular immunity in the immunized host (1, 34-36). Immuneresponses, especially antibody responses, are enhanced when the antigenis released into the extracellular environment as opposed to beingsequestered in the bacterial cytoplasm (11, 12).

These considerations led us to develop a RASV containment/deliverysystem capable of releasing antigen by cell lysis within the immunizedanimal. We utilized the tightly regulated araC P_(BAD)activator-promoter system to construct a strain/plasmid system thatdirects regulated arabinose-dependent, programmed lysis. Anarabinose-regulated cell lysis system should not be undermined byrelease into the environment, where stream and groundwater levels ofarabinose are in the sub-micromolar range (37). Studies in ourlaboratory have shown that in our Salmonella strains, P_(BAD) is notactivated by 13 μM (0.0002%) arabinose (S. Wang, personalcommunication).

We chose the asdA gene as the primary driver of cell lysis, since it isknown that, unlike some lethal mutations, a lack of Asd not only resultsin cell death, but also cell lysis (38). To further facilitatecontainment, we also included the murA gene in our scheme. The plasmidcopy of murA was derived from E. coli to reduce the potential forrecombination with the S. typhimurium chromosomal copy, a possibleescape strategy for the cell. Finally, we included the P22 P_(R)promoter driving transcription of anti-sense mRNA to silence anyresidual mRNA transcripts that may arise from the plasmid copies of asdAor murA in the absence of arabinose. In our system, the C2 repressor,which inhibits P22 P_(R) transcription, is only synthesized in thepresence of arabinose. Thus, in the arabinose-limiting environment inhost tissues, C2 is not made and anti-sense mRNA is transcribed.

The data of this example show that the system we have devised results incell lysis in the absence of arabinose and clearance of the strain fromhost tissues. More importantly, our strain was fully capable ofdelivering a test antigen and inducing a robust immune responsecomparable to that of a vaccine strain without this containment system,thereby demonstrating that this system has all the features required forbiological containment of a RASV. This plasmid-host system of regulateddelayed lysis in vivo depends on regulated delayed shut off in thesynthesis of enzymes essential for peptidoglycan synthesis results incomplete avirulence in the complete absence of any other attenuatingmutations. As such this is another means to confer a regulated delayedattenuation phenotype.

This system can be modified to suit a number of different needs forantigen delivery. We can add mutations that will delay lysis to allowadditional time for the RASV to colonize host tissues. For example, wehave deleted additional genes from the arabinose operon to preventarabinose metabolism, thereby maintaining an effective arabinoseconcentration in the cytoplasm for a longer period of time. Strains withthese arabinose gene deletions are currently being evaluated for use asantigen or DNA delivery vectors.

The regulated lysis system also has potential as a DNA vaccine vectordelivery system. An asdA deletion mutant of Shigella flexneri has beenused to deliver DNA in animals (39), but the immune responses were weak,presumably because the cells did not persist long enough to efficientlyinvade host tissues. A ΔasdA mutant of E. coli has also been used todeliver DNA in tissue culture (40). However, our system, whether usedfor Shigella, E. coli or Salmonella (41), provides the vaccine withadequate time to establish itself in host tissues before lysis occurs,thereby enhancing the probability of efficient DNA delivery.

Lastly, this system could be modified to provide effective biologicalcontainment for genetically engineered bacteria used for a diversity ofpurposes in addition to vaccines

REFERENCES FOR EXAMPLE 5

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(1998) A live recombinant avirulent oral    Salmonella vaccine expressing pneumococcal surface protein A induces    protective responses against Streptococcus pneumoniae. Infect.    Immun. 66: 3744-3751.-   23. Van Heijenoort J (1994) in Bacterial Cell Wall, eds Ghuysen J M,    Hackenbeck R (Elsevier, Amsterdam), pp 39-54.-   24. Black S, Wright N G (1955) Aspartic β-semialdehyde dehydrogenase    and aspartic β-semialdehyde. J. Biol. Chem. 213: 39-50.-   25. Brown E D, Vivas El, Walsh C T, Kolter R (1995) MurA (MurZ), the    enzyme that catalyzes the first committed step in peptidoglycan    biosynthesis, is essential in Escherichia coli. J. Bacteriol. 177:    4194-4197.-   26. Lee N L, Gieliw W O, Wallace R G (1981) Mechanism of araC    autoregulation and the domains of two overlapping promoters, P_(c)    and P_(BAD), in the L-arabinose regulatory region of Escherichia    coli. Proc. Natl. Acad. Sci. U.S.A. 78: 752-756.-   27. 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Example 6: Preparation of Vaccine Product

Master seed and working seed banks of each vaccine organism in separatevials have been prepared for frozen storage in vegetable-basedcryopreservative. Purity of the seed banks was established followingstandard operating procedures Full characterization of the seed banksincludes phenotypic evaluation on selective media, PCR, antigenicagglutination, colorimetric assays, LPS gel analysis, production ofcatalase to reveal the RpoS phenotype and demonstrated to reflect thecorrect and anticipated phenotype and genotype of the three vaccinestrains. Antibiotic sensitivity testing has confirmed that these strainsare sensitive to ciprofloxacin, ampicillin, ceftriaxone,trimethoprim/sulfamethoxazole (Table 15). Ampicillin, ciprofloxacin,ceftriaxone and trimethoprim/sulfamethoxazole are typically tested forminimum inhibitory concentrations (MICs) for Salmonella.

TABLE 15 Minimum inhibitory concentrations of antibiotics for RASV-Spstrains. Salmonella Typhi strain (μg/ml) Antibiotic χ9633(pYA4088)χ9639(pYA4088) χ9640(pYA4088) ampicillin <2 <2 <2 ciprofloxacin <0.25<0.25 <0.25 ceftriaxone <0.25 <1 <1 trimethroprim- <20 <20 <20 sulfa-methoxazole

The vials of vaccine Working Seed are maintained frozen in designatedboxes and entered into the freezers' inventory logs. The Working Seedvials are stored in duplicate freezers maintained between −65° and −75°C. Vaccine stability is determined by titration of representative vialsof each of the RASV-Sp Master and Working Seed banks at 0, 3, 6, 12, 24months and every 6 months thereafter. Table 16 shows the stability ofthe RASV-Sp Master Seed and Working Seed stocks as determined byquarterly viable titration.

TABLE 16 Stability of RASV-Sp Master Seed (MS) and Working Seed (WS)banks χ9633(pYA4088) χ9639(pYA4088) χ9640(pYA4088) Date of CFU/ml CFU/mlCFU/ml Titer MS WS MS WS MS WS Nov. 17, 2007 1.95 × 10¹⁰ 3.20 × 10¹⁰1.60 × 10¹⁰ 2.63 × 10¹⁰ 1.76 × 10¹⁰ 3.00 × 10¹⁰ Feb. 22, 2008 1.98 ×10¹⁰ 3.40 × 10¹⁰ 1.66 × 10¹⁰ 3.20 × 10¹⁰ 1.69 × 10¹⁰ 3.53 × 10¹⁰ May 17,2008 1.62 × 10¹⁰ 4.23 × 10¹⁰ 1.58 × 10¹⁰ 3.08 × 10¹⁰ 1.54 × 10¹⁰ 3.01 ×10¹⁰ Sep. 8, 2008 1.44 × 10¹⁰ 2.70 × 10¹⁰ 1.11 × 10¹⁰ 3.55 × 10¹⁰ 1.43 ×10¹⁰ 1.30 × 10¹⁰

Live, whole bacteria constitute the unformulated active immunogenicsubstance that when fermented in permissive conditions will beformulated with sterile PBS pH 7.4 to produce the final vaccine product.

The final vaccine products will be prepared on the day of administrationto the volunteers in the clinical trial to optimize immunogenicity andfitness of the strains.

Briefly, a 37° C. overnight culture of each vaccine strain is preparedfrom a frozen vial of RASV-Sp Working Seed. The next morning, thecultures are subcultured 1:20 into fresh, prewarmed media and shakengently at 37° C. to an optical density (OD) at 600 nm ideally between2.0-2.3. The cells are harvested by centrifugation and resuspendedgently in sterile PBS to the final dosage prescribed. Data collectedfrom production runs of the vaccine dosages conducted prior to the startof the clinical trial will be used to correlate the OD₆₀₀ of the finalPBS cell suspension to CFU/ml (GCGH-ASU-SOP-096-00, see CMC section ofthe IND application). This data will be used to confirm the target rangeof the final dosage prior to releasing the vaccine dosages to theclinic.

Table 17 shows the production record of three consecutive dosages of theRASV-Sp inocula for producing 10-ml final liquid dosages of live vaccinefor oral administration to adult volunteers. The data provide assurancethat the RASV-Sp vaccine inocula can be consistently produced within thetarget range of the dosage required on the start date of the clinicaltrial.

TABLE 17 RASV-Sp final dosage preparation record Hours to ProductionRASV-Sp Harvest culture Vaccine Dosage/ date Strain OD₆₀₀ harvest 10 ml¹Aug. 11, χ9633(pYA4088) 2.83 4 h 37 min 2.14 × 10⁷ 2008 χ9639(pYA4088)2.20 4 h 24 min 3.04 × 10⁷ χ9640(pYA4088) 2.38 4 h 2.30 × 10⁷ Aug. 19,χ9633(pYA4088) 2.11 3 h 58 min 1.14 × 10⁷ 2008 χ9639(pYA4088) 2.08 4 h40 min 2.22 × 10⁷ χ9640(pYA4088) 2.14 3 h 57 min 1.29 × 10⁷ Aug. 21,χ9633(pYA4088) 2.15 3 h 48 min 1.37 × 10⁷ 2008 χ9639(pYA4088) 2.01 4 h30 min 2.09 × 10⁷ χ9640(pYA4088) 2.14 3 h 42 min 1.51 × 10⁷ ¹Each lotproduced passed purity and identity testing following standard operatingprocedures.Formulation

The human fasting stomach can reach pH levels as low as 1.5. Low pHtolerance of the RASV-Sp strains was tested after suspending cells inmedium at pH 7, 4.5 or 3 for 1 hour at 37° C. Viability of the samplesafter incubation was assessed by plate counts. Data shown are theaverage number of CFU/ml recovered. In these studies, we included theparental wild-type S. typhi strains χ3744 (ISP1820), χ3769 (Ty2) andχ8438 (Ty2 RpoS⁺). We also included an attenuated S. typhi ISP1820strain (χ8110) that had been used in a previous trial in whichreactogenicity was observed. In all cases, the vaccine constructionsχ9633(pYA4088), χ9639(pYA4088) and χ9640(pYA4088) were more acidsensitive than their wild-type parents or than the attenuated ISP1820strain χ8110 (FIG. 44).

The PBS used as the diluent is unlikely to provide sufficient bufferingactivity. Since the stomach pH rises dramatically upon ingestion offood, we plan to increase the stomach pH of volunteers by administeringEnsure nutrition shakes prior to administering the vaccine dosages. FIG.45 shows the stability after one hour of the RASV-Sp vaccines suspendedin three different flavors of Ensure® nutrition shake.

Stability of RASV-Sp Strains

The RASV-Sp vaccine dosages maintain a stable titer suspended in the PBSat room temperature for a period of less than 2 hours. FIG. 46 showsthat the initial cell suspensions hold titers near 1×10¹⁰ CFU for up toan hour and then maintain stably after dilution in PBS for an additionalhour. The RASV-Sp final dosages will be administered to volunteerswithin two hours of resuspension in PBS to ensure optimalimmunogenicity.

Example 7: Nonclinical Studies

It should be noted that S. typhi is an obligate human pathogen and noanimal models are available for a full evaluation of the S. typhi-basedvaccines. Inoculation of newborn mice with high doses of wild-typevirulent strains of S. typhi, even when modified to express the S.typhimurium virulence plasmid needed by S. typhimurium to causedisseminated disease in mice, fails to infect or cause any signs ofdisease or any weight loss. We constructed, in parallel of theengineering of S. typhi, S. typhimurium strains bearing essentiallyidentical mutations as the S. typhi-based vaccines for pre-clinicalsafety and immunogenicity evaluation in mice.

Safety of S. typhimurium χ9558(pYA4088) in Newborn Mice.

A relevant safety test was to evaluate the safety in newborn and infantmice of S. typhimurium strain χ9558(pYA4088) [(Δpmi-2426 Δ(gmd-fcl)-26ΔP_(fur81)::TT araC P_(BAD) fur ΔP_(crp527)::TT araC P_(BAD) crpΔasdA27::TT araC P_(BAD) c2 ΔaraE25 ΔaraBAD23 ΔrelA 198::araC P_(BAD)lacl TT ΔsopB1925 ΔagfBAC811], which carries mutations nearly identicalto the S. typhi vaccine strains and the same plasmid to enable PspAexpression. Newborn mice are highly susceptible to wild-type S.typhimurium infection and succumb at oral doses lower than 100 CFU.

Newborn and infant mice were orally inoculated with 5 l containing1-3×10⁸ CFU of the strain χ9558(pYA4088) at 0, 2, 4 or 7 days of age.Table 18 shows the health status and survivors over a 10-week period. Nodisease symptoms or death occurred in any of the mice at any time afteroral inoculation with over 10⁶ times the wild-type LD₅₀.

TABLE 18 Safety of χ9558(pYA4088) in newborn/infant BALB/c mice Age ofHealth status mice Oral dosage 10 weeks post- Survivors/ (days) CFUvaccination total 0 1.0 × 10⁸ Healthy 9/9 2 1.2 × 10⁸ Healthy 12/12 43.0 × 10⁸ Healthy 11/11 7 3.5 × 10⁸ Healthy 13/13 The oral LD₅₀ for thewild-type parent strain χ3761 is less than 100 CFU.Distribution of S. typhimurium χ9558(pYA4088) in Tissues of Newborn Mice

Colonization of tissues from newborn and infant mice was evaluated 3 and7 days after oral inoculation with the S. typhimurium strainχ9558(pYA4088). Homogenized tissue samples from euthanized mice werespread onto agar plates and CFU/g enumerated. In addition, samples ofhomogenized tissues were also subjected to enrichment culture to revealpresence or absence of Salmonella. Table 19 shows the tissuedistribution of the attenuated S. typhimurium strain χ9558(pYA4088) innewborn mice to 7 days of age.

The levels of colonization of the intestinal tract by S. typhimuriumχ9558(pYA4088) were quite good. In this regard, it should be noted thatisolation of Peyer's patch tissue in these infant mice to determineSalmonella titers is not feasible. Titers in liver and spleen were lowerthan expected but this was interpreted as an indication of the safety ofχ9558(pYA4088) for newborn and infant mice.

These data in Table 16 and Table 17 show that the attenuated S.typhimurium vaccine strain with mutations nearly identical to the S.typhi vaccine strains is safe for newborn and infant mice. Therefore, itcan be extrapolated from these data that these mutations provide anequivalent level of safety to the S. typhi vaccines.

TABLE 19 Colonization data of χ9558(pYA4088) in tissues (CFU/gram) 3 and7 days post inoculation in infant mice Age of Oral Spleen LiverIntestine* Mice dosage Number (CFU/g) (CFU/g) (CFU/g) (day) (CFU) ofmice Day 3 Day 7 Day 3 Day 7 Day 3 Day 7 0 1.0 × 10⁸ 1 <10 5.9 × 10³ <106.8 × 10³ 2.7 × 10⁶ 6.3 × 10⁴ 2 <10 7.3 × 10³ <10 5.0 × 10⁴ 5.9 × 10⁵3.1 × 10⁵ 3 <10 2.4 × 10³ 3.0 × 10³ 2.5 × 10⁴ 1.6 × 10⁶ 2.4 × 10⁵ 2 1.2× 10⁸ 1 0 << 10 1.1 × 10³ 2.9 × 10³ 1.1 × 10³ 6.1 × 10⁵ 5.0 × 10⁵ 2 0 <<10 1.4 × 10³ 5.9 × 10² 1.7 × 10³ 2.3 × 10⁵ 5.4 × 10³ 3 2.5 × 10³ 1.7 ×10³ 5.7 × 10³ 3.3 × 10³ 2.7 × 10⁶ 3.1 × 10⁵ 4 3.0 × 10⁸ 1 3.3 × 10³ <105.2 × 10³ <10 1.1 × 10⁸ 5.4 × 10⁶ 2 <10 8.5 × 10³ 2.4 × 10³ 8.0 × 10³1.1 × 10⁸ 1.8 × 10⁷ 3 8.1 × 10⁴ 2.7 × 10³ 1.2 × 10⁴ 2.1 × 10⁴ 7.1 × 10⁶2.8 × 10⁷ 7 3.5 × 10⁸ 1 <10 <10 2.4 × 10² <10 7.0 × 10⁶ 1.5 × 10⁷ 2 <10<10 5.0 × 10² <10 1.1 × 10⁷ 6.0 × 10⁶ 3 <10 <10 3.2 × 10² <10 1.8 × 10⁷3.9 × 10⁶ *Entire small intestine and contentsEvaluation of Safety of S. typhi Vaccine Strains in Young Mice.

Newborn mice (<24 h) were each orally inoculated with 10 μl containing1×10⁹ CFU of each of the S. typhi vaccine strains. Table 20 shows thehealth status and survivors over a six-week period. No disease symptomsor death occurred in any of the mice at any time after oral inoculation.

TABLE 20 Safety of S. Typhi χ9633(pYA4088), χ9639(pYA4088) andχ9640(pYA4088) in newborn mice Health status Oral dosage 6-weeks post-Survivors/ Strain (CFU) inoculation total χ9633(pYA4088) 1.2 × 10⁹healthy 3/3 χ9639(pYA4088) 6.0 × 10⁸ healthy 3/3 χ9640(pYA4088) 7.5 ×10⁸ healthy 3/3Distribution of S. typhi Strains in Tissues of Newborn Mice.

Although S. typhi can invade murine cells with low efficiency (comparedto S. typhimurium), they do not survive well or multiply and quicklydecline in titer following oral administration. For this reason, theability of S. typhi to colonize (or not colonize) murine tissues is notnecessarily indicative of the ability of the strain to colonize humantissue. However, the distribution of S. typhi cells in tissues fromnewborn mice was evaluated as an addition to the data from the S.typhimurium RASV-Sp strain χ9558(pYA4088) (see Table 19).

Colonization was assessed 3 and 7 days after oral inoculation with theS. typhi vaccine and wild-type strains. The attenuated ISP1820 strainused in a previous trial (χ8110) and the typhoid vaccine strain Ty21 awere also included for comparative purposes. Homogenized tissue samplesfrom euthanized mice were spread onto agar plates and CFU/g enumerated.In addition, samples of homogenized tissues were also subjected toenrichment culture to reveal the presence or absence of Salmonella. FIG.47 shows the distribution of the S. typhi vaccine and wild-type strainsin the intestine, spleen and liver tissues 3 and 7 days afterinoculation. Data shown are the geometric means+standard deviations oftwo separate colonization experiments.

These data demonstrate that the mutant vaccine candidate S. typhistrains colonize mouse tissues no better than the wild-type parentalstrains. The additional strains Ty21 a and χ8110 showed similarly poorlevels of colonization. These results were not unexpected, since miceare unable to support an infection with S. typhi strains even wheninfected soon after birth.

Reactogenicity of PBS Diluent with and without S. typhi

The general safety test as directed in 21 CFR 610.11 was performed toaddress concerns raised of the possibility that residual mediacomponents might be reactogenic in volunteers.

The RASV-Sp PBS cell suspensions were filter-sterilized and thesecell-free solutions, along with sterile PBS and sterile growth mediumwere injected intraperitonneally into mice and guinea pigs. The weight,health and general well-being of study animals were monitored daily for7 days. At the conclusion of the study, animals were euthanized andnecropsied, and observable differences of the internal organs (includingalterations in size, shape, coloration and vascularization) werephotographed for comparative analysis.

All animals survived for the duration of the general safety test (7 daysafter injection). No unexpected or nonspecific responses were observedwith any of the RASV-Sp strains as compared to the PBS controls. Theaverage weights for each group throughout the course of the study areshown in FIGS. 48A and B. For each group, the animals weigh the same ormore on Day 7 than they did on the day of injection.

No diminishment of the health and general well-being, and no change inthe character of internal organs of mice and guinea pigs were noted.

These data provide evidence to support the conclusion that the traceamount of residual media components present in the final vaccinepreparation is unlikely to be reactogenic in human volunteers.

Immunogenicity Assessment of S. pneumoniae Antigen

The immunogenicity of the PspA antigen of S. pneumoniae was assessedusing the Asd⁺ plasmid vector pYA3634. The pYA3634 plasmid is aprecursor of pYA4088 and encodes aa 3-257 of the PspA-Rx1 protein(pYA4088 spans aa 3-285) (See FIG. 49). Cultures of the RASV-Sp strainsgrown in the presence of arabinose synthesize the Lacl repressor at highlevels to repress transcription from P_(trc) on the Asd⁺ plasmid vectorpYA3634 to minimize synthesis of PspA until after immunization when thevaccine strain is already colonizing internal lymphoid tissues. 0.05%arabinose and 0.2% mannose were used to prepare S. typhimuriumχ9558(pYA3634) (Δpmi-2426 Δ(gmd-fcl)-26 ΔP_(fur81)::TT araC P_(BAD) furΔP_(crp527)::TT araC P_(BAD) crp ΔasdA27::TT araC P_(BAD) c2 ΔaraE25ΔaraBAD23 ΔrelA 198::araC P_(BAD) lacl TT ΔsopB1925ΔagfBAC811) toevaluate relative IgG response to PspA-Rx1 expressed from χ9558(pYA3634)in BALB/c mice compared to χ9088(pYA3634) (ΔP_(fur33)::TT araC P_(BAD)fur Δpmi-2426 Δ(gmd-fcl)-26 ΔasdA33) and χ8133(pYA3634) (Δcya-27 Δcrp-27ΔasdA16). Groups of 7-week-old female BALB/c mice were orallyadministered approximately 10⁹ CFU of each strain and boosted with thesame dose at 8 weeks. Blood was obtained by mandibular vein puncturewith heparinized capillary tubes at biweekly intervals. ELISA wasperformed to determine IgG antibody titers to PspA, S. typhimurium LPS.FIG. 50 shows total serum IgG titers to the PspA protein and to S.typhimurium LPS.

Four weeks after the second oral immunization, mice were challenged intwo experiments with approximately 5×10⁴ CFU of S. pneumoniae WU2. Bothexperiments gave similar results, and the data have been pooled forpresentation and analysis. This challenge dose resulted in the deaths of100% of the unvaccinated mice, with a mean time to death of 2-3 days.

The percent protection rate and the number of days of survival afterchallenge with virulent S. pneumoniae strain WU2 are shown in FIG. 51.Seventy-one percent of the mice immunized with χ9558(pYA3634) wereprotected from pneumococcal challenge. This is significantly higher thanthe level of protection observed for the Δcya Δcrp strain χ8133(pYA3634)(p=0.0063).

Passive Transfer of Pneumococcal Immunity.

An experiment to demonstrate passive-antibody transfer of protectiveimmunity to pneumococcal challenge was conducted in mice. Mice wereorally inoculated with 1×10⁹ CFU of a RASV-Sp strain containing eitherthe empty vector pYA3493 or the vector pYA3634 and boosted with the samestrain and dose 8 weeks after primary immunization. At week 12, serafrom immunized mice were collected and pooled.

Naïve, syngeneic BALB/c mice received 100 l in the tail vein ofundiluted serum from pooled serum of immunized mice. All groups werechallenged intraperitoneally 12 h later with S. pneumoniae WU2. Thepercent survival of mice receiving pooled serum was assessed 15 daysafter challenge with S. pneumoniae WU2. Table 21 shows the percentsurvival of mice that were protected by passive-antibody transfer fromchallenge with more than 250 LD₅₀ doses of the virulent S. pneumoniaeWU2.

Sera from mice immunized with S. typhimurium χ9558(pYA3634) passivelyprotected 100% of mice challenged with over 250 LD₅₀ doses of thevirulent S. pneumoniae WU2.

TABLE 21 Passive transfer of pneumococcal immunity by serum from donorsimmunized with S. Typhimurium vaccines expressing PspA Volume of %survival the donor of Strain No. serum (μl) pooled Donors immunized withexpresses of administered serum vaccine strain PspA mice IV recipients¹Saline control — 5 100 0 χ8133(pYA3493) No 5 100 0 Δcya-27 Δcrp-27ΔasdA16 χ9088(pYA3493) No 5 100 0 Δpmi-2426 Δ(gmd-fcl)-26 ΔP_(fur81)::TTaraC P_(BAD) fur ΔasdA33 χ9558(pYA3493) No 5 100 0 Δpmi-2426Δ(gmd-fcl)-26 ΔP_(fur33)::TT araC P_(BAD) fur ΔP_(crp527)::TT araCP_(BAD) crp ΔasdA27::TT araC P_(BAD) c2 ΔaraE25 ΔaraBAD23 ΔrelA198::araCP_(BAD) lacI TT ΔsopB1925 ΔagfBAC811 χ8133(pYA3634) Yes 5 100 80χ9088(pYA3634) Yes 5 100 100 χ9558(pYA3634) Yes 5 100 100 ¹Mice werechallenged IP 12 h after receiving donor immune serum with >250 LD₅₀doses of S. pneumoniae WU2Immunogenicity of χ9633(pYA4088), χ9639(pYA4088), and χ9640(pYA4088) inFemale 6- to 7-Week-Old BALB/c Mice.

The ability of the S. typhi RASV-Sp strains administered intranasally toBALB/c to induce serum antibody titers to PspA was assessed(GCGH-ASU-SOP-074-00, see CMC section of the IND application). Mice wereinoculated intranasally with 10 μl of approximately 10⁹ CFU of a RASVstrain with either the empty vector pYA3493 or the PspA⁺ vector pYA4088.Sera were collected 2, 4, 6 and 8 weeks after vaccination and anti-PspA,-LPS and -OMP IgG titers determined by ELISA.

It should be noted that this type of immunogenicity assay has been usedby others even though we believe it is of marginal value. This isbecause S. typhi (wild-type or mutant) is unable to successfully invadeand persist in murine cells or lymphoid tissues as is S. typhimurium.FIGS. 52A-C show the total IgG response to PspA, LPS and OMP from seracollected over an 8-week period after intranasal administration of theRASV strains with the PspA plasmid pYA4088 or the empty vector pYA3493.All RASV strains harboring either pYA3493 or pYA4088 equally inducedsignificant anti-LPS and anti-OMP IgG titers as soon as two weekspost-inoculation. PspA IgG titers gradually increased over theeight-week period from mice administered the RASV-Sp strains. Althoughthe group size was small, the RASV-Sp Ty2 RpoS⁺ strain χ9640(pYA4088)induced a slightly higher anti-PspA IgG titer than the ISP1820derivative χ9633(pYA4088).

Complement Deposition Assay and Passive Protection of Mice Using Serumfrom Human Vaccine Volunteers.

Sera from the vaccine volunteers which test positive for PspA will beevaluated for their ability to passively protect mice from pneumococcalinfection. Passive transfer of protective immunity to pneumococcalchallenge will be demonstrated by transfer of pre- and post-immune serumand the antibodies it contains to naive unimmunized mice followed byintravenous challenge with virulent S. pneumoniae.

As an additional measure of the protective capacity of the anti-PspAresponse in volunteers, sera may be further evaluated by the complementdeposition assay. This test will quantitatively evaluate the ability ofantibody in pre- and post-immune sera to facilitate deposition ofcomplement C3 onto S. pneumoniae. Immunization of humans with PspA hasbeen shown to lead to elevated levels of antibody to PspA, increases inthe ability of the sera to mediate complement deposition on S.pneumoniae, and increases in the ability of human sera to protect micefrom fatal pneumococcal infection. The deposition of complement on S.pneumoniae has been shown to correlate inversely with the ability of S.pneumoniae to cause invasive disease.

Example 8: Non-Clinical Assessment of Safety

Additional safety tests were conducted to address concerns raisedregarding the apparent lack of adequate safety data for the ISP1820derivative strain χ9633(pYA4088). Another ISP1820 derivative, χ8110Δcfs),(Δcya-27Δcrp-pabA-40 Acts), was shown to be safe in Phase Iclinical trials. To bridge the previous human data with χ8110 to thepresent vaccine candidate χ9633(pYA4088), additional safety data weregenerated to demonstrate that χ9633(pYA4088) is equivalent to or moreattenuated than χ8110 as evaluated by survival in human blood andperipheral blood monocytes. Comparisons to the Ty21 a vaccine Vivotif®which is the gold standard for live Salmonella vaccine safety were alsoincluded in the following non-clinical assessment of safety.

Survival of RASV-Sp Strains in Human Blood

The bactericidal effects of heat-treated and untreated whole blood werecompared by incubating the RASV-Sp strains and wild-type S. typhicounterparts in the presence of normal whole blood (GCGH-ASU-SOP-081-01,see CMC section of the IND application).

Approximately 1×10⁶ CFU of each RASV-Sp strain, χ8110, Ty21a and theirwild-type counterparts were added to duplicate 1.5 ml blood aliquotsfrom volunteers. Blood was collected in accordance with the ASU humanuse protocol #0804002872. Survival of the Salmonella strains was assayedin blood that had been heat inactivated (HI) by incubation at 55° C. forone hour prior to inoculation, or in untreated, active (A) blood.Viability of the Salmonella strains was measured by plating samples onpermissive media 0, 3, 6 and 18 hours after inoculation. FIG. 53 showsthe geometric mean of the CFU recovered of at least 3 trials±thestandard deviation.

The RASV-Sp candidates are severely attenuated in their ability tosurvive in whole human blood as compared to wild-type S. typhi andχ8110. Vaccine strain levels drop below the threshold of detectionwithin 3 hours and the strains did not regrow at the later timepoints ofthe assay. This is in contrast to χ3744, χ3769 and χ8110, which are notonly present at significantly higher levels, but also replicate in theblood at the later timepoints of the assay. The RASV-Sp candidates,including the ISP1820 derivative χ9633(pYA4088), are as attenuated asTy21 a and more attenuated than the ISP1820 RASV χ8110 used in aprevious clinical trial.

Sensitivity of RAS V-Sp Strains to Native Guinea Pig Serum Complement.

The bactericidal properties of guinea pig serum complement weredetermined for the RASV-Sp strains and their wild-type counterparts.Guinea pig complement was used for this assay because of its high levelof bacteriocidal activity.

The S. typhi strains χ3744 (wild-type ISP1820), χ3769 (wild-type Ty2),χ8438 (RpoS⁺ wild-type Ty2), χ9633(pYA4088), χ9639(pYA4088) andχ9640(pYA4088) were prepared following GCGH-ASU-SOP-062-01 Preparationof RASV-Sp dosages for adult volunteers. The sensitivity of the cells tocomplement was assayed following GCGH-ASU-SOP-091-00 Resistance ofRASV-Sp strains to guinea pig complement. Strains were assayed in PBSonly, complement (purified from guinea pig serum) only, and complementwith anti-S. typhi O-antigen D₁ opsonizing antibody. Reactions wereincubated for 3 hours at 37° C., and then the viability of theSalmonella strains was measured by plating on permissive media. Datashown in FIG. 54 represent the average CFU/ml.

Both the wild-type Salmonella typhi strains and the RASV-Sp strains aresensitive to killing by complement in the presence of Salmonella typhiO-antigen specific D₁ antibody. The vaccine strains are killed to amoderately higher degree than the wild-type strains. In the absence ofS. typhi-specific antibody, the wild-type strains are resistant tocomplement-mediated killing. However, the RASV-Sp strains exhibit a highlevel of sensitivity to complement-mediated killing even in the absenceof opsonizing antibody.

Survival of RASV-Sp Strains in Peripheral Human Mononuclear Cells.

Rubin et al. demonstrated that in patients with typhoid fever,circulating S. typhi cells are associated with mononuclear cell-plateletfraction of whole blood. Because this serovar does not typically causedisease in mice or other animals, the development of rapid ex-vivoassays using freshly elutriated peripheral blood mononuclear cells(PBMCs) have been demonstrated as reliable tools for determiningattenuation of S. typhi for vaccine research and development.

PBMCs derived from blood of 3 different volunteers were elutriatedfollowing GCGH-ASU-SOP-082-01 Survival of RASV-Sp strains in peripheralhuman mononuclear cells. After incubation of PBMCs and bacteria in24-well culture plates for 1, 3 and 23 additional hours, PBMCs werelysed and cell lysates were plated onto permissive media to determineviable CFU. Survivability of the RASV-Sp strains χ9633(pYA4088),χ9639(pYA4088) and χ9640(pYA4088) compared to χ8110 (ISP1820 Δcya-27Δcrp-pabA-40 Δcfs), Ty21 a and to wild-type S. typhi χ3744 (wild-typeISP1820), χ3769 (wild-type Ty2), χ8438 (RpoS⁺ wild-type Ty2) are shownin FIGS. 55A-C. The data shown are the geometric means+standarddeviations of three separate assays.

The peripheral blood mononuclear cell assay used to measure the invasionand persistence of the S. typhi strains readily distinguished betweenvirulent S. typhi and the attenuated RASV-Sp strains and Ty21a, known tosurvive poorly both in vitro and in vivo. The wild-type Ty2 and ISP1820strains invaded and persisted at a significantly higher rate than theRASV-Sp strains and Ty21a (p<0.05).

Both χ9639(pYA4088) and Ty21 a were the least fit to survive and persistin PBMCs compared to the wild-type Ty2 RpoS⁻ strain (p=0.0022 and 0.0022at 24 hours, respectively), which may be a consequence of possessing therpoS mutation. These results are consistent with the RpoS⁻ phenotype inthat null mutants are susceptible to killing by macrophage and exhibitincreased sensitivity to environmental stress.

The ISP1820 derivative χ9633(pYA4088) was equivalent to χ8110 insurviving within PBMCs at 2, 4 and 24 hours (p=1.00, 0.505 and 0.878,respectively) and both strains were significantly reduced in theirability to invade and persist within PBMCs compared to the wild-typeISP1820 at all timepoints.

Together these data demonstrate further safety of the RASV-Sp strains.Additionally the ability of the ISP1820 derivative χ9633(pYA4088) toinvade to a lesser degree than the wild-type ISP1820 but persist at alow level in PBMCs demonstrates that this strain is not compromised toreach host target cells to deliver the PspA for antigen processing.

Taken collectively, the RASV-Sp strains are adequately attenuated due totheir extreme sensitivity to complement-mediated killing, their poorsurvival in whole human blood and in fresh elutriated peripheral bloodmononuclear cells. The ISP1820 derivative χ9633(pYA4088), althoughsufficiently attenuated by the data presented here, may display the bestattributes for antigen presentation to the appropriateantigen-presenting cells of the host immune system.

RASV-Sp Shedding and Survival in Human Stool

A consequence of oral administration of live Salmonella vaccineorganisms is that they can be shed transiently in the stool of vaccinerecipients. An important aspect of the potential impact of environmentalrelease of a live vaccine is to evaluate the duration, rate of sheddingand the survival rate. Endeavors to develop live vaccines that reduceshedding have been met with variable success. The licensed live oraltyphoid vaccine, serovar Typhi Ty21 a, is shed at low rates in thestools of most vaccines for 1 to 4 days. Ideally, it is desirable tolimit the number of genetically modified microorganisms entering theenvironment, without decreasing vaccine immunogenicity or efficacy.

An initial assessment of the duration of shedding following oralinoculation was conducted in 6-week old adult mice. The S. typhi RASV-Spstrains χ9633(pYA4088), χ9639(pYA4088), and χ9640(pYA4088), the S.typhimurium RASV-Sp counterpart χ9558(pYA4088) and the S. typhiwild-type strains χ3744, χ3769 and χ8438 were grown. Approximately 1×10⁹CFU of each strain was administered orally to groups of 3 mice. Sheddingwas monitored for 6 days after inoculation by homogenizing fecal pelletsand plating on selectively differential media. The data shown in Table22 represent the average number of CFU/ml detected for each group. Noneof the S. typhi strains (wild-type or RASV-Sp) were detected more than 3hours following the inoculation. The S. typhimurium RASV-Sp strainχ9558(pYA4088) was also not detected after the initial day ofinoculation. These data indicate that significant levels of vaccineorganism shedding are confined to the initial day of immunization.Low-level shedding (less than 10³ CFU/ml) may occur for a slightlylonger period.

TABLE 22 Fecal shedding of RASV-Sp strains and S. Typhi wild-typestrains following oral inoculation of mice. 3 Hours 18 Hours Day 2 Day 4Day 6 (CFU/ (CFU/ (CFU/ (CFU/ (CFU/ Strain ml) ml) ml) ml) ml)χ9558(pYA4088) 1.7 × 10⁷ <10³ <10³ <10³ <10³ χ3744 1.7 × 10⁷ <10³ <10³<10³ <10³ χ9633(pYA4088) 8.0 × 10⁶ <10³ <10³ <10³ <10³ χ3769 1.0 × 10⁷<10³ <10³ <10³ <10³ χ9639(pYA4088) 1.6 × 10⁷ <10³ <10³ <10³ <10³ χ84381.8 × 10⁷ <10³ <10³ <10³ <10³ χ9640(pYA4088) 1.6 × 10⁶ <10³ <10³ <10³<10³ Limit of detection for this assay was 10³ CFU/ml

Since S. typhi is unable to efficiently attach to and invade to theintestinal epithelial cells of mice, the results of the previous studymay not accurately represent the duration of shedding from a human host.In order to gather data about the competitive fitness of the strains inthe human intestinal environment, the RASV-Sp and wild-type S. typhistrains were evaluated in anaerobic human stool samples. Viability ofthe S. typhi strains was assessed by plating dilutions onto selectivemedia 1, 3, 7 and 10 days after inoculation of fresh stool suspensionswith approximately 1×10⁸ CFU/ml. Inoculated samples were incubated at37° C. in an anaerobic environment. The limit of detection forrecovering the S. typhi strains was 10⁴ CFU/ml.

FIG. 56 shows the survival of the S. typhi wild-type χ3744 (wild-typeISP1820), χ3769 (wild-type Ty2), χ8438 (RpoS wild-type Ty2) and RASV-Spstrains χ9633(pYA4088), χ9639(pYA4088) and χ9640(pYA4088) in human stoolover the 10-day period of evaluation. The RASV-Sp strains were notrecoverable 24 hours after inoculation of the stool samples and remainedbelow the threshold of detection (10⁴ CFU) throughout the remainder ofthe study. The wild-type strains, however, persisted through day 3 atmeasurable titers above 10⁴ CFU and then fell below the level ofdetection through day 10 of the study.

These data represent the worst case scenario as the RASV-Sp strains wereprepared in this study to allow the regulated-delayed expression of thenear wild-type attributes that would endow the strains withcharacteristics that would make them most fit for survival. In reality,once ingested by volunteers, the strains will eventually lose and nolonger display these protective attributes due to the absence ofexogenous arabinose and mannose and would rapidly succumb to the harshand competitive environment present in stool.

Survival of S. typhi in Canal Water, Chlorinated Water and Sewage

The aim of this study was to compare the survivability of the RASV-Spstrains and S. typhi wild-type counterparts in several conditions thatmimic the environment and to address concerns regarding the impact ofreleasing live attenuated, genetically-modified organisms into theenvironment.

Three environmental conditions were prepared to serve as test materialfor assessing survivability of the S. typhi strains. Chlorinated waterwas prepared to contain approximately 3 to 5 ppm chlorine. The S. typhitest strains were washed twice to remove residual media andapproximately 1×10⁸ CFU of each strain were added to triplicate tubescontaining the test solution. Raw sewage was retrieved from a localwaste water treatment plant in Phoenix, Ariz. Untreated canal water wascollected from the Phoenix metropolitan area. Viability of the S. typhistrains was assessed by plating dilutions onto selective media 1, 3, 7and 10 days after inoculation of the triplicate test solutions withapproximately 1×10⁸ CFU/ml. The limit of detection for recovering the S.typhi strains was 10⁴ CFU/ml.

FIG. 57A-C shows the survival of the S. typhi wild-type χ3744 (wild-typeISP1820), χ3769 (wild-type Ty2), χ8438 (RpoS wild-type Ty2) and RASV-Spstrains χ9633(pYA4088), χ9639(pYA4088) and χ9640(pYA4088) in theenvironmental test solutions. The RASV-Sp and wild-type strains wereextremely sensitive to chlorinated water experiencing several logs ofkilling after a 30-minute exposure (FIG. 57A). The RASV-Sp strains wereless fit than the wild-type strains to persist in canal water decreasingmore than 3 logs in titer over the 10-day evaluation period (FIG. 57B).Titers of the RASV-Sp strains in raw sewage dropped steadily decreasingmore than 3 logs in titer over the 10-day period (FIG. 57C). These datashow that the RASV-Sp strains did not display any enhanced attributes tosurvive in these environmental test solutions over the Ty2 or ISP1820wild-type strains.

In summary, the data show that the RASV-Sp strains do not have acompetitive advantage in chlorinated water, untreated surface water orsewage over naturally-occurring organisms and are no more likely topersist in these conditions than the wild-type Salmonella typhi.

Example 9. Response to S. typhi Vaccines in Adult Mice Immunized byIntranasal Response

Immune Response to S. typhi Vaccines in Adult Mice Immunized byIntranasal Response.

Adult BALB/c mice (7 weeks) were inoculated intranasally withapproximately 1×10⁹ CFU of RAStyV strains carrying either rPspAexpression plasmid pYA4088 or control plasmid pYA3493 in 10 μl, andboosted with the same dose of the same strain six weeks later. Sera werecollected 2, 4, 6 and 8 weeks after vaccination and serum IgG responsesto rPspA, S. typhi LPS and S. typhi OMPs were measured by ELISA. Thisexperiment was performed twice, with each group (8 mice) receivingapproximately the same dose of vaccine. Sera from all mice in a groupwere pooled for analysis. Absorbance levels of a secondary anti-mouseantibody conjugated to HRP was recorded at 405 nm using an automatedELISA plate reader (model EL311SX; Biotek, Winooski, Vt.). Absorbancereadings that were 0.1 higher than PBS control values were consideredpositive. The results from both experiments were similar and have beenpooled for analysis.

Results:

All mice immunized with strains expressing pspA developed anti-PspAantibodies (FIG. 58A). Anti-PspA titers were boosted after the secondimmunization at 6 weeks. Strain χ9640(pYA4088) (Ty2 RpoS⁺) induced asignificantly higher anti-rPspA IgG titer in mice than those of eitherthe ISP1820 derivative χ9633(pYA4088), or the Ty2 derivativeχ9639(pYA4088) at all time points (P<0.01). After boosting, theanti-rPspA IgG antibody levels in χ9639(pYA4088) immunized mice weresignificantly higher than the mice immunized with χ9633(pYA4088)(P<0.05). No anti-PspA IgG was detected in mice immunized with PBS orstrains carrying pYA3493.

All RAStyV strains induced significant anti-LPS titers (FIG. 58B) andOMPs (FIG. 58C) as early as two weeks post inoculation. After the secondimmunization, significant boosting of serum antibody responses to LPSand OMPs was observed (P<0.01).

Mucosal IgA anti-PspA responses were slow to develop, but reached hightiters after boosting (FIG. 58D).

Protection of Adult Mice Immunized with S. typhi Vaccines AgainstChallenge with Virulent S. pneumoniae.

Method: At week 10, mice were challenged by intraperitoneal injectionwith 1.0×10⁴ CFU of S. pneumoniae WU2 (50 LD₅₀) in 100 μl BSG.Challenged mice were monitored daily for 30 days.

Result: All mice immunized with three S. typhi vaccine strainsexpressing pspA were significantly protected compared with controls(FIG. 41). The protection afforded by the Ty2 derivatives, χ9639(pYA4088) and χ9640 (pYA4088) was significantly greater than that theprotective effects of χ9633 (pYA4088) (**, P<0.01).

Example 10. Comparative Phase I Protocol to Test Safety andImmunogenicity in Adult Volunteers of Three Recombinant AttenuatedSalmonella typhi Vaccine Vectors Producing Streptococcus pneumoniaeSurface Protein Antigen PspA

This trial was conducted in compliance with the protocol, InternationalConference on Harmonisation Good Clinical Practice E6 (ICH-GCP) and theapplicable Food and Drug Administration and other Department of Healthand Human Services regulatory requirements. Study design is summarizedbelow and in FIG. 59.

Objectives:

Objective 1.

To evaluate maximum safe tolerable single dose levels of the threerecombinant attenuated S. typhi vaccine vectors (χ9639(pYA4088) S. typhiTy2 RpoS⁻, χ9640(pYA4088) S. typhi Ty2 RpoS⁺ and χ9633(pYA4088) S. typhiISP1820) using dose escalation studies in healthy adult volunteers.

Objective 2.

To evaluate immunogenicity of the three recombinant attenuated S. typhivaccine vectors [χ9639(pYA4088) S. typhi Ty2 RpoS⁻, χ9640(pYA4088) S.typhi Ty2 RpoS⁺ and χ9633(pYA4088) S. typhi ISP1820] with regard totheir abilities to induce mucosal and systemic antibody and cellularimmune responses to the S. pneumoniae PspA antigen and to Salmonella LPSand outer membrane protein (OMP) antigens.

Study Outcome Measures

Primary Outcome Measures:

Safety and tolerability will be measured by assessment of reactogenicityand Adverse Events following vaccination. Escalation to the next doselevel will occur only after review of the safety data from day 28post-inoculation of the previous Arm.

Secondary Outcome Measures:

Immunogenicity testing will include antibody and/or cellular responsesto vaccine at Days 0, 7, 28, 84 and 168.

Hypotheses Tested

The recombinant attenuated χ9639(pYA4088) S. typhi Ty2 RpoS⁻,χ9640(pYA4088) S. typhi Ty2 RpoS⁺ and χ9633(pYA4088) S. typhi ISP1820vaccine vectors will be safe when given orally to healthy adult humanvolunteers.

The χ9640(pYA4088) S. typhi Ty2 RpoS+ recombinant attenuated vaccinevector will induce higher titers of antibodies to the Streptococcuspneumoniae PspA antigen than will the parental χ9639(pYA4088) S. typhiTy2 RpoS⁻ vector.

The χ9633(pYA4088) S. typhi ISP1820 recombinant attenuated vaccinevector will induce higher titers of antibodies to the Streptococcuspneumoniae PspA antigen than will either the parental χ9639(pYA4088) S.typhi Ty2 RpoS⁻ or χ9640(pYA4088) S. typhi Ty2 RpoS⁺ vaccine.

Study Design

The study was a dose escalating study divided into four Arms (1-4). EachArm will consist of 3 groups (A-C) of 5 healthy young adults 18-40 yearsof age and each group (A-C) will be administered one of three differentvaccine vectors. Each subject will receive an oral dose of vaccine onday 0 and be followed closely to determine the safety, tolerability andimmunogenicity of the vector. The vaccine vector found to be both safeand immunogenic with maximum immunogenicity and ease of geneticmanipulation will be selected as the parent for second generationvaccine vectors to deliver multiple S. pneumoniae protective antigens.

Arm 1 will evaluate the attenuated strains of χ9639(pYA4088) S. typhiTy2 RpoS⁻, χ9640(pYA4088) S. typhi Ty2 RpoS⁺ and χ9633(pYA4088) S. typhiISP1820 in an initial single oral dose (10⁷ CFU), evaluating safety andimmunogenicity of the recombinant attenuated strains. An escalation indose will proceed only after demonstrating the safety and tolerabilityof the lower vaccine dose through Day 28.

Arm 2 will evaluate an escalation of dose (10⁸ CFU) for safety andimmunogenicity in 3 groups of 5 new volunteers. An escalation dose willproceed only after demonstrating the safety and tolerability of thelower vaccine dose through Day 28.

Arm 3 will evaluate an escalation of dose (10⁹ CFU) for safety andimmunogenicity in 3 groups of 5 new volunteers. An escalation dose willproceed only after demonstrating the safety and tolerability of thelower vaccine dose through Day 28.

Arm 4 will evaluate an escalation of dose (10¹⁰ CFU) for safety andimmunogenicity in 3 groups of 5 new volunteers. This is the highest doseto be tested

The dose escalation schedule is provided below:

TABLE 23 Vaccination Schedule Vaccine Groups and Dose A B C (n = 5/χ9639(pYA4088) χ9640(pYA4088) χ9633(pYA4088) group) Ty2 RpoS⁻ Ty2 RpoS⁺ISP1820 Arm 1 10⁷ CFU 10⁷ CFU 10⁷ CFU Arm 2 10⁸ CFU 10⁸ CFU 10⁸ CFU Arm3 10⁹ CFU 10⁹ CFU 10⁹ CFU Arm 4 10¹⁰ CFU  10¹⁰ CFU  10¹⁰ CFU 

The study will enroll Arms 1 through Arms 4 in succession as data arereviewed following each Arm and the Safety Monitoring Committee (SMC)authorizes the next Arm to enroll based on review of 28-day safety dataincluding final blood and stool culture results obtained from previousArm. This review cycle allows for an interval of a minimum of 35 days ofreview of all data from the current Arm, after enrollment of the lastsubjects in the current Arm, before proceeding to the next higher dosageArm of the study.

Maximum Limit of Tolerability and Dose Escalation of a Specific Strain

Escalation to the next dose level of any of the three vaccine vectorswill occur only if the safety data in the preceding dose level cohortfor a specific vaccine are acceptable to the SMC and the PI. Escalationto higher dose levels for each of the three vaccines shall proceed inthis manner until the highest dose level is reached, or dose-limitingtoxicity (maximum limit of tolerability) prevents further doseescalation. Dose escalation of a specific strain shall not proceed inthe event that: 3 or more individuals within 1 dose level develop thesame severe laboratory abnormality and the abnormality is deemedmedically significant by the SMC and is determined to be associated withvaccine; or if 2 or more individuals develop a severe systemic reactionthat is determined to be associated with the vaccine; or if 1 individualdevelops an SAE determined to be associated with vaccine.

Subject Selection Criteria

Volunteers will be healthy 18-40 year old male or non-pregnant femaleadults who fully understand the purpose and details of the study.Subject exclusion criteria include history of Salmonella infection orvaccination, and a history of pneumococcal vaccine.

What is claimed is:
 1. A recombinant bacterium capable of regulatedexpression of at least one nucleic acid sequence encoding an antigen ofinterest, wherein the bacterium comprises: a. at least one chromosomallyintegrated nucleic acid sequence encoding a repressor operably linked toa regulatable promoter, wherein the nucleic acid sequence encoding therepressor and/or promoter have been modified from the wild-type nucleicacid sequence so as to optimize the expression level of the nucleic acidsequence encoding the repressor; b. a vector comprising at least onenucleic acid sequence encoding an antigen of interest operably linked toa promoter regulated by the repressor, such that the expression of thenucleic acid sequence encoding the antigen is repressed during in vitrogrowth of the bacterium, but the bacterium is capable of high levelexpression of the nucleic acid sequence encoding the antigen in a host;and c. the mutation ΔrelA197::araC P_(BAD) lacl TT.
 2. The recombinantbacterium of claim 1, wherein the bacterium further comprises a deletionin asd.
 3. The recombinant bacterium of claim 1, wherein the bacteriumfurther comprises the mutation ΔasdA::TT araC P_(BAD) c2.
 4. Therecombinant bacterium of claim 1, wherein the repressor is selected fromthe group consisting of Lacl, C2, and C1.
 5. The recombinant bacteriumof claim 1, wherein the nucleic acid sequence encoding the repressorcomprises a modified Shine-Dalgarno sequence and optimized codons so asto optimize the expression level of the nucleic acid sequence encodingthe repressor.
 6. The recombinant bacterium of claim 1, wherein thevector is a plasmid.
 7. The recombinant bacterium of claim 1, whereinthe nucleic acid encoding the antigen of interest is operably linked toa P_(trc) promoter.
 8. The recombinant bacterium of claim 1, wherein theantigen of interest is toxic.
 9. The recombinant bacterium of claim 1,wherein the vector further comprises a nucleic acid sequence encoding asecretion signal for the antigen of interest.
 10. The recombinantbacterium of claim 1, wherein the bacterium is capable of eliciting aprotective immune response in the host.
 11. The recombinant bacterium ofclaim 1, wherein the bacterium comprises more than one means ofattenuation.
 12. The recombinant bacterium of claim 1, wherein therepressor is Lacl; the regulatable promoter is selected from the groupconsisting of P_(rhaBAD), P_(xyIAB), and P_(xyIFGH); the vector is aplasmid; and the nucleic acid sequence encoding the antigen of interestis operably linked to the P_(trc) promoter.
 13. A vaccine compositioncomprising the recombinant bacterium of claim
 1. 14. A method foreliciting an immune response in a host, the method comprisingadministering to the host an effective amount of a vaccine compositioncomprising a recombinant bacterium of claim 1.